Abstract: A new medium soya nutri nuggets extract medium with different glucose concentration for lactic acid bacteria is presented. In this we used soya nutri nuggets extract, which is supported good growth for lactic acid bacteria isolated from milk and milk products and glucose, which affects cell growth and bacteriocin biosynthesis. For many of the strains tested, bacteriocin production in Soya Nutri Nuggets Extract Medium (SNNEM) were similar to those achieved in MRS medium. This medium allowed growth to continue to higher densities than four other defined media. SNNEM is compared with MRS and other previously published medium for lactic acid bacteria.
INTRODUCTION
Lactic Acid Bacteria (LAB) are anaerobic Gram-positive bacteria with a GRAS (Generally Regarded as Safe) status. They are also food grade bacteria and therefore, they can be used for the delivery of proteins of interest in foodstuff or in the digestive tract. Lactic acid bacteria is widely distributed in milk and milk products as a part of the naturally accurring microflora and as deliberately added starter cultures (Hammes and Knauf, 1994) their most important habitat is in untreated milk, fermented milk and cheeses. Lactococcus lactis subsp. lactis, either in pure form or associated with other micro-organisms is the mesophilic strain most commonly used as a starter culture for lactic products, thus they fulfill an irreplaceable role in ensuring the structure, taste, conservation and healthfulness of these products (Salminen and Von Wright, 1993; Boonmee et al., 2003; Ziadi et al., 2005; Do-Won et al., 2006).
One of the important steps in the study of bacteriocins is their production. The composition of culture medium and culture conditions such as temperature, pH and time of incubation have profound effect on the production of bacteriocins. In general, conditions that provide high cell density favour high bacteriocin concentration.
It is necessary to study the physiological conditions most suited for their production. For examination of nutritional requirements a defined must be used as such a medium (i) provides reproducibility of chemical composition, (ii) avoids an unnecessary excess of nutrients, allowing the determination of nutrients limitation, (iii) meets the experimentally determined nutritional requirements of several strains and (iv) supports growth at a reasonably high rate (Foucaud et al., 1997).
Lactococcus are extremely fastidious organism with numerous growth requirements. They are usually grown in a complex medium such as MRS (De Man et al., 1960). However, such media are too expensive for an economical production process. Recently, there has been increased interest in the production of bacteriocin from soya permeate since certain LAB strains can grow and produce appreciable amounts of bacteriocin in this medium, which has a low cost (De Vuyst and Vandamme, 1994).
The purpose of our investigation was therefore to develop an inexpensive basal medium from soya nutri nuggets extract to produce a high bacteriocin titre using Lactococcus lactis CCSUB202 and evaluate the effect of aeration and controlled pH on bacteriocin production using soya nutri nuggets extract medium.
MATERIALS AND METHODS
Bacterial Strains and Media
The bacteriocin producing strain used in this study was Lactococcus
lactis CCSUB202. Bacteriocin bioactivity was tested quantitatively
with Lactococcus lactis subsp. lactis MTCC 3038
as a test organism. Strains were maintained on MRS slant bottles at 4°C
for shorter duration of 1 to 2 months. For longer duration, the cultures
were stored frozen at -20°C in 30% sterile skimmed milk in small vials
of 2 mL.
Soya Nutri Nuggets Extract Medium (SNNEM) was supplemented with various nutrients such soya nutri nuggets extract (1000 mL), glucose (40 g), sodium acetate (3.0 g), KH2PO4 (2.0 g), MgSO4 (0.2 g), Tween-80 (1 mL). The medium was inoculated and incubated at 37°C for 24 h to study their effect on bacteriocin production in the developed medium. Optimization of physical factors (pH, Temperature and Incubation period) for bacteriocin production on soya nutri nuggets extract medium.
Fermentation
A 24 h old culture of actively growing cells was always used as the
inoculum; the inoculum size was always 10% (v/v). The fermentation was
carried out in a 2 L bench fermenter (LAB FORS AG CH-4103 Bottmingen/Switzerland)
with pH, temperature, dissolved oxygen, agitation and aeration control,
using 1 L of medium composition, aeration and control pH on bacteriocin
production has been evaluated. The samples were withdrawn at regular intervals
to determine the concentration of total protein concentration, pH and
bacteriocin activity.
Growth Determination
Growth (biomass) was measured by determination of turbidity after
withdrawn the sample take O.D. (optical density) by spectrophotometer
(Systronics UV-VIS double-beam spectrophotometer 2201) at 650 nm.
Protein Estimation
Protein concentration was determined by the method of Lowry et
al. (1951) using Bovine Serum Albumin (BSA) as standard.
Bacteriocin Activity Determination
Inhibitory activity titres against the indicator bacteria were determined
by agar diffusion well assay with slightly modification. Fresh culture
of bacteriocin sensitive strain (Lactococcus lactis subsp. lactis
MTCC3038) was inoculated in 40 mL of sterilized MRS broth and incubated
at 37°C for 24 h. One hundred microliter of broth culture was spread
on fresh MRS medium plates. Wells were cut with sterile cork borer (4
mm in diameter).
Fifty microliter of CFF (cell free filtrate) which was serially diluted 1:2, 1:4,1:8, 1:16, 1:32, 1:64, 1:128, 1:256, 1: 512 (Two fold dilution) was placed in each well with the help of micropipette using sterilized tips. The plates were then incubated at 37°C for 24 h, without inversion. After incubation the plates were observed for clear circular zones of inhibition around the wells. The diameter of zone of inhibition was measured in mm with the help of standard scale (Zone measured scale, Hi-media). The highest dilution that gave a well defined zone of inhibition of growth was used to calculate AU mL-1 (Highest dilution that showed a distinct zone of inhibition X 20 (1000 μL/50 μL).
| AU mL-1 = highest dilution that showed clear well-defined zone of inhibition x 1000 μL/Volume (μL) used in the well. |
RESULTS AND DISCUSSION
Mass Production of Bacteriocin in SNNEM
Bacteriocin production in SNNEM by batch fermenter (LAB FORS AG CH-4103
Bottmingen/Switzerland) with a capacity of 2 L was used in present study.
The relationship between absorbance and bacteriocin activity by Lactococcus lactis subsp. lactis CCSUB202 as influenced by temperature and pH values was assessed. Table 4 represent fermentation at a controlled temperature 37°C and at constant pH 7.5 after optimized the physical parameters Table 1-3.
Table 4 shows that the bacteriocin production by Lactococcus lactis
subsp. lactis CCSUB202 in a batch fermenter was slightly increased
as compared to fermentation in shaker culture. In this experiment the
highest bacteriocin activity was reached at the end of the exponential
growth phase 5280 AU mL-1 of medium (Table 4) when absorbance
had also reached a maximum value in experiment. Bacteriocin activity was
already detectable after 4 h of fermentation when 40% of the absorbance
had been produced. Table 4 shows that maximum activity was obtained at
the 10th h of fermentation (5280 AU mL-1). However, the higher
absorbance was achieved in this experiment at 10th h of fermentation Table 4.
| Table 1: | Bacteriocin production from Lactococcus lactis subsp. lactis CCSUB202 in SNNEM broth at 37°C at different pH |
| SNNEM = Soya Nutri Nuggets Extract Medium | |
| Table 2: | Bacteriocin production from Lactococcus lactis subsp. lactis CCSUB202 in SNNEM broth at different incubation temperatures |
| SNNEM = Soya Nutri Nuggets Extract Medium | |
| Table 3: | Bacteriocin production by Lactococcus lactis subsp. lactis CCSUB202 in SNNEM broth at 37°C at different incubation periods |
| SNNEM = Soya Nutri Nuggets Extract Medium | |
| Table 4: | Bacteriocin production in batch fermenter at 150 rpm at constant temperature (37°C) and pH 7.5 in SNNEM |
| SNNEM = Soya Nutri Nuggets Extract Medium | |
Similar behaviour was observed by De Vuyst and Vandamme (1992) using Lactococcus lactis subsp. lactis NIZO22186 and glucose as a carbon source. These experiments achieved 1.27 g L-1 of cell mass in the eighth hour of fermentation and nisin activity of approx. 2000 IU mL-1 of medium. Several other workers Callewaert et al. (1999), De Vuyst et al. (1996) and Lejeune et al. (1998) also used fermenter for bacteriocin production.
CONCLUSION
Lactococcus lactis subsp. lactis CCSUB202 produced bacteriocin in SNNEM (Soya Nutri Nuggetes Extract Medium), which showed higher bacteriocin production than other used medium.