Effect of Prebiotics on Lactobacillus acidophilus Growth and Resulting pH Changes in Skim Milk and a Model Peptone System

The effect of Raftiline® GR, Raftiline® HP-Gel, and Raftilose® P95 prebiotics on growth curves of the probiotic Lactobacillus acidophilus LA-K and resulting pH changes in skim milk and a model system consisting of 0.1% peptone was investigated. Skim milk (100 mL) and 0.1% peptone (100 mL) each containing either 1% or 3% Raftiline® GR, Raftiline® HP-Gel, or Raftilose® P95 prebiotics were autoclaved, cooled to 37°C, and inoculated with 0.132 mL of L. acidophilus LA-K. Both the growth of L. acidophilus and the changes in pH were followed for 16 h at 37°C. The growth was determined by measuring the optical density (turbidity) at 600 nm. Prebiotics had a greater influence on the growth of L. acidophilus and a greater pH lowering effect in a peptone model system than in skim milk. This observation seems logical since more nutrients are available in skim milk compared to peptone. The system, skim milk or peptone, in which prebiotics are used, does influence the overall desirable effect of the prebiotic. Effect of Prebiotics on Lactobacillus acidophilus Growth and Resulting pH Changes in Skim Milk and a Model Peptone System


Introduction
Probiotics have been defined as "live microorganisms which when administered in adequate amounts confer a health benefit on the host" [1]. Probiotics include many different species, especially species of the genera Lactobacillus and Bifidobacterium [2]. A commonly used probiotic bacterium is L. acidophilus.
Health benefits provided by probiotics include improving gastrointestinal health [3], modulating immune function [4], possibly helping to prevent colon cancer [5], possibly mediating an antihypertensive effect [6], reducing urinary tract infections in women [7], improving lactose digestion [8], reducing serum cholesterol levels [9], and possibly preventing many other health problems. The probiotic properties of survival in an in vitro gastro-intestinal tract model system and adhesion to Caco-2 cells have been shown for L. acidophilus LA5 [10].
Gibson et al. [11] defined prebiotics as "a selectively fermented ingredient that allows specific changes, both in the composition and/ or activity in the gastrointestinal microflora that confers benefits upon host well-being and health". Prebiotic intake has been reported to be effective against bowel cancer, inflammatory bowel disease, pathogenic agents, coronary heart disease, necrotizing enterocolitis, and obesity and can improve mineral availability. Insulin and oligofructose, transgalacto-oligosaccharides, and lactulose can be classified as prebiotics [11]. Inulin and oligofructose are found in many types of plants as plant storage carbohydrates [12]. Inulin has a linear chain consisting of either α-D-glucopyranosyl-[ß-D-fructofuranosyl] n-1 -ß-D-fructofuranoside or a ß-D-fructopyranosyl-[ß-D-fructofuranosyl] n-1 -ß-D-fructofuranoside [11]. The fructosyl-glucose linkage is ß(2↔1) and the fructosyl-fructose linkages are ß(1←2). Chicory inulin has a degree of polymerization ranging from 2 to approximately 60. Oligofructose (fructo-oligosaccharides) is obtained by partial enzymatic hydrolysis of inulin with endo-inulinase (EC 3.2.1.7) and has a degree of polymerization ranging from 2 to 7 with an average of 4 [11]. Oligofructose and inulin that are commercially available have variable chain lengths. The prebiotics used in the present study have a degree of polymerization of 4 (P95) (a short chain oligofructose) [13], an average degree of polymerization of at least 10 (GR) (a medium chain inulin) and a degree of polymerization greater than 23 (HP-Gel) (a long chain inulin) [14]. Prebiotic chain lengths influence various characteristics. Rats had greater bioavailability of calcium upon consumption of long chain fructan (inulin) compared to short chain oligofructose [15]. In contrast with medium and long chain fructooligosaccharides, the short chain fructo-oligosaccharides are the first to be consumed by bifidobacteria resulting in greater lactate and acetate production [16].
Products that contain both probiotic cells and prebiotic substances are called synbiotic products. Inulin and oligofructose can be used as an ingredient in various products including probiotic yogurt [17] and probiotic ice cream [18]. Araůjo et al. [19] developed a cottage-like synbiotic cheese that contained Lactobacillus delbrueckii UFVH2b20 and inulin and this product maintained a microorganism count high enough allowing it to be considered a probiotic throughout its shelf life. Consuming synbiotic yogurts containing Bifidobacterium animalis subsp. lactis and inulin can increase bifidobacterial numbers and decrease clostridial numbers in some people [20].
Milk is an excellent medium for microbial growth and hence it is heavily regulated and has legal limits for bacteria [21]. Peptone (0.1%) is often used to make dilution blanks to enumerate microorganisms [22,23]. It is not known if prebiotics would have a greater beneficial effect on growth of L. acidophilus in a system devoid of milk such as in common, but it can be labor intensive. Turbidity is a rapid procedure, but it does not distinguish between viable and dead cells. Fluorescent dye staining has been successfully used [24] to measure bacterial viability.
The objective of the present study was to compare the 16 hour growth curves of L. acidophilus and resulting pH changes in skim milk and in a model system consisting of 0.1% peptone each containing either 1% or 3% oligofructose or inulin with various chain lengths.

Materials
Bacto™ peptone (Becton, Dickinson and Company, Sparks, MD, USA) was obtained from University Stores at Louisiana State University. Raftiline® GR, Raftiline® HP-Gel, and Raftilose® P95 prebiotics were obtained from BENEO-Orafti (Oreye, Belgium). L. acidophilus LA-K was obtained from Chr. Hansen (Milwaukee, WI, USA) and had a total cell count of greater than 9x10 9 cfu/g in the pure culture (communication with the manufacturer).

Sample preparation
For each replicate, skim milk and 0.1% peptone were each poured into five milk dilution bottles to a volume of 100 mL. Three types of prebiotics (Raftiline® GR, Raftiline® HP-Gel, and Raftilose® P95) at either 1% or 3% (w/v) were individually added to skim milk and 0.1% peptone in milk dilution bottles. One set of controls for both skim milk and 0.1% peptone contained L. acidophilus but no prebiotics while the other set of controls contained neither prebiotics nor L. acidophilus. The treatments and controls for the skim milk and 0.1% peptone were autoclaved at 121°C for 15 minutes and cooled to 37°C in an incubator. An aliquot of 0.132 mL of L. acidophilus LA-K was added to each treatment and one controls each for the skim milk and 0.1% peptone. Three replicates for both the 1 and 3% prebiotic levels were performed.

Growth curves
Immediately after addition of L. acidophilus LA-K to each sample, a 3 mL aliquot was aseptically taken from the milk dilution bottle and placed into a disposable cuvette for preparation of the growth curve. Cuvettes were kept within the 37°C incubator for 16 hours except for analysis once every 40 minutes. Growth curves were prepared by measuring the optical density (turbidity) at 600 nm in a disposable cuvette with a Nicolet Evolution 100 spectrophotometer (Thermo Electron Corporation, Madison, WI, USA).

pH measurements
Milk dilution bottles containing the samples and the control used for determining the pH were kept within the 37°C incubator for 16 hours except for analysis once every 40 minutes. The pH was measured with an Ultra Basic pH/mV meter (Denver Instrument Company, Arvada, CO, USA). The pH meter was standardized with pH 4 and 7 buffers before use.

Optical density of skim milk containing prebiotics
The optical densities of skim milk containing 1% prebiotics and their controls are presented in figure 1. For samples containing 1% prebiotics, there was typically less than 0.1 difference in optical densities of samples containing L. acidophilus. The optical densities of the P95 containing samples tended to be slightly higher than the optical densities of the remaining samples and the control containing L. acidophilus. The optical densities of skim milk containing 3% prebiotics and their controls are shown in figure 2. It appeared that L. acidophilus LA-K entered the stationary phase after about 12 hours of incubation for samples containing either 1 or 3% prebiotics. Although there were larger differences in optical densities among samples containing 3% prebiotics compared to samples containing 1% prebiotics, slopes of the optical densities versus incubation time curves for all of the samples and the control containing L. acidophilus still appeared to be approximately similar. The rate of growth of L. acidophilus in all of the L. acidophilus containing samples and the control containing L. acidophilus appeared to be approximately similar making it difficult to show the stimulating effect of these prebiotics within 16 hours of measurement. However, it is possible that there could have been a growth stimulating effect by the prebiotics later in the stationary phase or in the death phase that occurred beyond the approximate 16 hours of measurement.
Growth curves of L. acidophilus have been reported in the literature. In a recent study regarding L. acidophilus LA-K homogenized at various pressures, it was found that this culture stayed in the logarithmic phase of growth for at least 10 hours of incubation [25]. Liong    reported that various strains of L. acidophilus and Lactobacillus casei had rapid growth for the first 9 to 15 hours of incubation and then slower growth afterwards to 24 hours when monitored by measuring absorbance at 620 nm and grown in a medium without cholesterol.
L. acidophilus can utilize various oligosaccharides and polysaccharides. All of the tested strains of L. acidophilus (33200, 837, DDS-1, and NCFM) were able to grow on an MRS-fructooligosaccharide agar [27]. L. acidophilus originating from swine feces degraded (arabino) galactooligosaccharides and xylooligosaccharides, partially degraded arabinogalactan enriched polysaccharide fraction and fructooligosaccharides, but did not degrade arabinan enriched polysaccharide fraction, arabinooligosaccharides, rhamnogalacturonan enriched polysaccharide fraction, rhamnogalacturonooligosaccharides, galacturonooligosaccharides, arabinoxylan polysaccharide from wheat flour, and arabinoxylooligosaccharides [28]. L. acidophilus NCFM can utilize isomaltooligosaccharides [29]. L. acidophilus DSM 20079 was able to grow on MRS media containing pectin or inulin since these counts of L. acidophilus were nearly as high as counts when grown on MRS media containing glucose after 24 hours [30].
Variable results have been reported as to the effect of prebiotics on the growth of various strains of L. acidophilus and L. acidophilus 4461 did not have higher counts in reconstituted skim milk containing 1, 2, or 3% Raftiline HP compared to reconstituted skim milk without Raftiline HP after 6 hours of incubation at 37°C [31]. When fermenting reconstituted skim milk with L. acidophilus LAC4 and S. thermophilus TA040, increased L. acidophilus counts were obtained when reconstituted skim milk was supplemented with 4% (w/w) lactulose compared to reconstituted skim milk without lactulose [32] and with maltodextrin, polydextrose, and oligofructose added individually compared to reconstituted skim milk without these prebiotics. Russo et al. [33] showed that the simultaneous availability of D-glucose and exopolysaccharides stimulated the growth of L. acidophilus NCFM by extending the logarithmic phase and delaying its entrance into the stationary phase. They also found that L. acidophilus reached a maximum cell concentration after 24 hours and a death phase after 30 hours when grown in a chemically defined medium supplemented with D-glucose. Nazzaro et al. [34] found that inulin did not affect the growth of L. acidophilus DSM 20079 compared to its growth in glucose. However, Saminathan et al. [35] found large differences in specific growth rates in representative Lactobacillus species in media supplemented with different oligosaccharides. Variations in specific growth rates with different strains of L. acidophilus show the importance of using the proper type of prebiotics while preparing synbiotics.

Optical density of 0.1% peptone containing prebiotics
The L. acidophilus growth curves are presented in figure 3 when measured in 0.1% peptone containing 1% prebiotics and their controls and in figure 4 when measured in 0.1% peptone containing 3% prebiotics and their controls. Compared to the curves in Figure 1 and 2, the curves in Figure 3 and 4 had lower starting and ending OD values. This was caused by the peptone model system being devoid of casein micelles that are naturally present in skim milk which would increase OD values. The optical densities usually decreased with incubation time, and this may have been due to the growth curve being in the death phase. The decrease was slower for the peptones that contained prebiotics compared to the control. This observation was probably due to slower death of L. acidophilus in the peptones containing prebiotics compared to the control. The greater effectiveness of prebiotics in stimulating L. acidophilus growth in 0.1% peptone compared to in skim milk may be explained by more nutrients being available for L. acidophilus in the skim milk compared to peptone, thereby reducing the need for additional nutrients provided by the prebiotics.

pH of skim milk containing prebiotics
The pH values of skim milk containing 1% prebiotics and their controls are presented in Figure 5. Although the pH values of the P95-containing samples tended to be slightly lower than the pH values of the remaining samples, the pH values of all of the samples containing prebiotics and the control containing L. acidophilus were very similar to each other. This similarity indicated that the prebiotics did not appear to have a major effect on rate of decrease of pH by L. acidophilus in skim milk. The pH values of skim milk containing 3% prebiotics and their controls are shown in figure 6. Likewise, there was not much difference in rate of decrease of pH values in the skim milks that contained different types of prebiotics. The decrease in pH over time results from the breakdown of lactose to form lactic acid [36]. The lack of differences in pH of the treatments compared to the control may be caused by the buffering capacity of skim milk arising from the caseins, whey proteins, phosphate and citrate.
In addition to growth, variable results have also been reported as to the effect of prebiotics on the decrease in pH of various strains of L. acidophilus. The pH did not drop faster in reconstituted skim milk containing 1, 2, or 3% Raftiline HP than in reconstituted skim milk without Raftiline HP after 6 hours of incubation with L. acidophilus 4461 at 37°C [31]. Supplementing reconstituted skim milk that was fermented with L. acidophilus LAC4 and S. thermophilus TA040 with 4% (w/w) lactulose increased the lactic acid acidity after day 1, day 7, and day35, increased the maximum acidification rate, decreased the time to reach the maximum acidification rate and the time to reach pH 4.5, and decreased the pH after 7 and 35 days [32]. When skim milk was supplemented with maltodextrin, oligofructose, and polydextrose individually, similar effects as with lactulose supplementation for lactic acid concentration, maximum acidification rate, and time to reach pH 4.5 were also found [37].

pH of 0.1% peptone containing prebiotics
The pH values of 0.1% peptone containing 1% prebiotics and their controls are presented in Figure 7. The pH values of the samples containing prebiotics were lower than the pH value of the control sample containing L. acidophilus. Both the GR-and HP Gel-containing samples had early decreases in pH and lower pH values than the P95containing samples. It did appear that prebiotics stimulated the rate of decrease of pH in 0.1% peptone. The pH values of 0.1% peptone containing 3% prebiotics and their controls are shown in Figure 8. Although there was an initial decrease in pH for the control that did not contain the L. acidophilus, this decrease in pH was smaller than the decrease in pH for the GR-containing and HP-Gel-containing samples. The P95-containing samples generally had less variation than both the control containing L. acidophilus and the GR-containing and HP-Gel-containing samples. Therefore, the GR-containing and HP-Gel-containing samples, but not P95-containing samples, were effective in stimulating the growth of L. acidophilus. There was a distinct pH-lowering effect in peptone compared to skim milk because peptone is a simple system and not as nutritionally dense as skim milk. Kunová et al. [38] showed that inulin was usually the most effective for stimulating the growth of various lactobacilli measured after 24 hours of incubation.

Conclusion
Prebiotics had a greater influence on the growth of L. acidophilus and a greater pH lowering effect in a peptone model system than in skim milk. The system, skim milk or peptone, in which prebiotics are used, does influence the overall desirable effect of the prebiotic.