Keywords: CC 85-92, leaves of sugar cane, pH control, substrate concentration.

Lactococcus are coccibacteria, which form chains of variable length, they have a homo-fermentative metabolism and produce exclusively L(+) lactic acid (Roissart, 1994), although Akerberg et al. (1998) report that, D(-) lactic acid. can be produced at low pH. Lactococcus lactis is sub-divided into other sub-species: lactis and cremoris (Schleifer and Kilpper-Balz, 1987; Kim et al. 1999). Lactococcus lactis, originated from the lactic Industrial products, have been, and are still being, extensively studied for their commercial potential, but that strain isolated from plants has been given less attention (Martínez-Cuesta et al. 1997); in sugar mills, Lactococcus lactis, like many other lactic acid bacteria, are involved in sucrose inversion, due to their potential for producing lactic acid. However, the commercial potential of these strains that are well adapted to sucrose-rich environments has not been researched, and there are no studies in the scientific literature in which lactic acid production is quantified for each of the strains involved in sucrose inversion. Representatives of this genus are isolated mainlyfrom fresh vegetables and from the skin of animals (Roissart, 1994). They also play an important role in aroma enhancement, the production of flavored milks, and in milk and cheese flavorings, and recently a great deal of attention has been focused on their pro-biotic properties (Salminen and Von Wright, 1993; Boonmee et al. 2003). The major product of fermentation is lactic acid, a compound with a high commercial value, with applications in the food, cosmetic, medical, and pharmaceutical industries. (Boonmee et al. 2003). However, the most interesting application for the lactic acid is its potential in the production of biopolymers (Hujanen and Linko, 1996; Chang et al. 1999; Hujanen et al. 2001; Danner et al. 2002; Lee, 2005). This abstract presents the results obtained from fermentations using glucose and a strain of Lactococcus lactis subs lactis isolated from sugar cane plants and also the effect of substrate concentration and pH control on lactic acid concentration (AL), conversion of sugar (CG) and yiel (Y_p/s).

Samples were taken from 12.3 month-old sugar cane plants, of the CC85-92 variety, and from sugar cane juice, at La Cabaña farm and sugar refinery (Cauca, Colombia). Each of the samples was diluted sufficiently to obtain isolated colonies, using 0.1% peptone water, and each was planted in duplicate in MRS agar (de Man et al. 1960). The medium was inoculated with 0.1 ml of each of the dilutions and they were incubated at 36º and 45ºC for 48 hrs in anaerobic conditions. Once the pure cultures were obtained, they were transferred to MRS liquid culture medium, and incubated under the same conditions described above. After 24 hrs the pure liquid cultures were centrifuged at 5000 g for 10 min and then filtered with Millipore HVLP02500 filters; the supernatants were then injected into an HPLC.

The strains that produced more than 12 gl^-1 of lactic acid under the described conditions were stored in MRS culture medium with glycerol, and they were frozen for preservation and later use. The homo-fermentative strain which produced the highest lactic acid concentration was selected. For this investigation, 18 fermentation trials were carried out at 32ºC, using MRS broth (250 ml). In order to study the effect of pH control, fermentations were carried out without pH control, and with pH controlled at 6.0. The effect of substrate concentration was studied by varying the glucose concentration in MRS broth, between 20, 60 and 110 grams per litter.

Analytical method

The strain was biochemical identified by duplicate, using API 50 CHL. The isomeric identification of the produced acid was made by enzymatic analysis D-lactic acid / L- lactic acid. The identification of the glucose metabolism by homofermentative or heterofermentative pathway was made using high-efficiency liquid chromatography (HPLC), quantifying the concentration of alcohol and acetate in MRS broth after the fermentative process. Sugars and lactic acid concentrations were measured using HPLC. Biomass was calculated as 540 nm from optical density data, using a spectrum-photometer. CG was calculated using initial glucose concentration (gl^-1) and final glucose concentration (gl^-1). Yp/s in grams per gram was calculated as the gradient of the curve obtained by plotting product concentration against substrate concentration.

20 strains isolated from sugar can plan were selected for production of lactic acid, of which only one homo-fermentative strain produced significant quantities (12.4 g l^-1 of lactic acid at 36ºC and 13.7 gl^-1 at 32ºC). None of the strains produced significant quantities of lactic acid at 45ºC. The strain was biochemical identified as Lactococcus lactis subs lactis.The strain produced lactic acid with the isomeric configuration L(+).

The variance analysis showed that glucose concentration and pH control have a highly significant effect on lactic acid concentration, glucose conversion and product yield (P < 0.005) with S values of 0.4457, 0.7696 and 0.02321 respectively. The interaction graphic showed a considerable interaction between pH and glucose concentration upon lactic acid concentration and yield and less marked interaction effect occurred for the glucose conversion. The kinetic parameters, calculated from the experimental data, can be seen in Table 1, while the kinetics of lactic acid production and of substrate consumption can be seen in Figure 1 and Figure 2 respectively.

The highest lactic acid concentration was achieved with 60 gl^-1 of glucose and with pH control. It is also important to highlight the good performance of the strain at low glucose concentrations (20 gl^-1) without pH control, in terms of lactic acid production and yield. The highest Y_p/s  were obtained at low glucose concentrations and without pH control, while the largest percentages of CG were obtained with low glucose concentrations, but with pH control. The significant concentration of lactic acid obtained in 48 hrs of fermentation with 20 gl^-1 of glucose, and without pH control (13.7 gl^-1) can be explained by the adaptation of the strain to environments with high sucrose concentrations and besides because it has been demonstrated that Lactococcus lactis ssp. lactis is capable of surviving and replicating in more extreme levels of stress, compared to other lactic acid strains (Kim et al. 1999). This is why the microorganism has an enzymatic system that hydrolyzes the disaccharide and then metabolizes the glucose using the glycolytic pathway; the behavior of this isolation in substrates rich in sucrose was studied by Serna-Cock and Rodriguez-de Stouvenel (2004). The lower lactic acid concentration obtained using 20 gl^-1 and pH control is due to an evident limitation of substrate.

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