Lactic acid bacteria are usually used as starter cultures in food technology are known to manufacture antimicrobial products having immense potential. Our study aims to isolate potential strain of lactic acid bacteria which has a potential for lactic acid production and to characterize it and to optimize culture conditions for the process. For this we have isolated a total of 38 strains of lactic acid bacteria (LAB) from dairy effluent samples that were collected from OMFED dairy plant of Rourkela, Orissa. The isolated strains were identified on basis of their morphological, physiological and biochemical characteristics. The distribution of the isolates by genus was as follows: Staphylococcus, Lactobacillus, Brevebacterium Streptococcus, Leuconostoc and Lactococcus. The colonies, which showed zone of clearance in starch agar plates, were maintained on to MRS agar plate for further experiments. Quantification of Lactic acid conversion from starch into Lactic acid by the selected microbial isolates was carried out.
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Dairy industry produces huge volumes of wastes, both solids and liquids. This waste poses escalating disposal and pollution (High BOD or COD) problems and represents a loss of valuable biomass and nutrients. However, despite of their pollution and hazard aspects, in many cases, dairy processing wastes have a good potential for conversation into useful products of higher value as by-product, or even as raw material for other industries. Organic acids are examples of such valuable by-product of the fermentation of high carbohydrate containing industrial substrates. They therefore could be utilized cheaply as substrate for microorganisms producing intermediate volume high value organic acids like lactic acid. Lactic acid is under increasing demand in Food, Pharmaceutical and Chemical Industries and for production of Poly lactic acid polymers, which possess outstanding biomedical applications. The global manufacture of this organic acid is estimated to be 100 million pounds/yr and is expected to grow by 8.6% annually (Narayanan et. al, 2004.) Lactic acid is currently manufactured either through chemical or by microbial fermentation mode. In India, the annual production capacity of lactic acid is 6000 T and an estimate gap of 2300 T in supply by the year 2015 have been predicted, if the present level of production is not increased (TIFAC 2001). Wastes generated from dairy plants may be regarded as a viable option for meeting this growing demand for lactic acid, if appropriate biotechnological interventions are used. Specific sectors amongst the Indian dairy manufacturing need to be targeted.
Lactic acid bacteria are a group of related bacteria that produce lactic acid as a result of carbohydrate fermentation. These microbes are broadly used by us in the production of fermented food products, such as yogurt (Streptococcus spp. and Lactobacillus spp.), cheeses (Lactococcus spp.), sauerkraut (Leuconostoc spp.) and sausage. These organisms are heterotrophic and usually have complex nutritional necessities because they lack many biosynthetic capabilities. Most species have multiple requirements for amino acids and vitamins. Because of this, lactic acid bacteria are generally abundant only in communities where these requirements can be provided. They are often associated with animal oral cavities and intestines (eg. Enterococcus faecalis), plant leaves (Lactobacillus, Leuconostoc) as well as decaying plant or animal matter such as rotting vegetables, fecal matter, compost, etc. Lactic acid bacteria are used in the food industry for several reasons. Their growth lowers both the carbohydrate content of the foods that they ferment, and the pH due to lactic acid production. It is this acidification process which is one of the most desirable side-effects of their growth. The pH may drop to as low as 4.0, low enough to inhibit the growth of most other microorganisms including the most common human pathogens, thus allowing these foods prolonged shelf life. The acidity also changes the texture of the foods due to precipitation of some proteins, and the biochemical conversions involved in growth enhance the flavor. The fermentation (and growth of the bacteria) is self-limiting due to the sensitivity of lactic acid bacteria to such acidic pH.
Lactic acid bacteria are amongst the best-studied microorganisms for human health advantageous effects and for fermentation. Significant novel developments have been made in the research of lactic acid bacteria in the areas of multidrug resistance, bacteriocins, osmoregulation, autolysins and bacteriophages. Advance has also been made in the production of food grade genetically modified Lactic acid bacteria. These have opened new potential applications for these microorganisms in various industries (12). In the previous studies, LAB could be isolated from many kinds of sources, such as milk products, fermented food, animal intestines or freshwater fishes (7), soils in vineyards (6), sugar cane plants (13), poultry farms in Senegal (4) but studies on the isolation of LAB from dairy effluents remain scarce. The desirable uniqueness of these microorganisms for industrial use is their ability to rapidly and completely ferment cheap raw materials, requiring minimal amount of nitrogenous substances. In turn providing high yields of preferred stereo specific lactic acid under conditions of low pH and high temperature, production of low amounts of cell mass and negligible amounts of other byproducts. Addition of lactic acid aqueous solution to the packaging of poultry and fish increases their shelf life (1). Therefore dairy isolates of lactic acid bacteria capable of degrading dairy effluent and converting them to Lactic acid are considered to be key to the development of a workable microbial fermentation based value addition process for dairy wastes containing Lactic acid bacteria.
Materials and Methods
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Characterization of dairy effluent samples: Dairy effluent samples from dairy plant of Rourkela, Orissa, India were collected in sterile containers. Physiochemical characters like conductivity, pH, BOD, COD, Total solids, etc were measured. Conductivity of the sample was measured by conductivity meter. The pH of the effluent samples was determined with an ion-specific electrode. The experimental parameters determined namely COD, TSS, TDS, and pH were estimated in accordance with the standard methods (2)
Isolation of Lactobacillus bacteria from dairy effluent: Lactobacillus sp. was isolated by serial dilution method from dairy effluent. The media containing Dextrose, Peptone and NaCl was prepared and sterilized. 0.1ÂÂ ml of the serially diluated dairy effluent samples was plated on nutrient agar media. The petriplate was placed inside the incubated at 30oC. After 24 hr, the colonies which showed zone of clearance in nutrient agar plates, were maintained on to MRS agar plate for further experiments.
Preparation of liquid Medium and culture condition: Nutrient medium (Dextrose 5g/L, Peptone 7.5g/l and Nacl 2.5 g/L) was prepared using distilled water and sterilized at 1210C for 15 min at 15 psi. The pH was adjusted to 6.5. The sterilized medium was inoculated with 1 ml of seed culture and incubated at 370C and 200 rpm in shaker incubator for overnight to carry out further experiments.
Morphological and Biochemical tests: Based on the growth performance in nutrient agar, lactic acid strains were selected. The isolates were identified based-on morphological and biochemical characterization according to Bergeyââ‚¬â„¢s Manual of Systematic Bacteriology (14). A single potential strain was selected for future experiments capable of producing lactic acid by utilizing dextrose as substrate.
Optimized condition for lactic acid production and estimation: The optimum temperature and pH for the Lactobacillus strain were determined by growing culture at various temperature and pH. For temperature optimization the bacteria was cultured in a temperature range of 260C-400C while for pH optimization, the pH range of 4.5-9.5 was used. Various carbon sources like (Dextrose, Sucrose and Starch soluble) in the form of saccharides and disaccharides were used for observing lactic acid production. Mediums were inoculated with 5 ml of overnight grown fresh seed Lactobacillus isolate in each case. Lactic acid concentration was estimated in each culture sample titrimetrically at a regular interval according to the protocol followed by Association of Official Analytical Chemists, A.O.A.C., 1990. To optimize % inoculum size/volume various inoculum sizes ranges from 1% to 5% were added in the fresh medium. After 12 and 24 hr of culture period, optical density of all samples were measured at 570 nm in a Systronics 2203 double beam spectrophotometer.
Lactic acid estimation Protocol: Concentration of lactic acid in sample was determined by method described earlier. Briefly, liquid broth was taken in centrifuge tubes and centrifuged at 8000 rpm for 15 minutes to pellet out the bacterial cells. The supernatant was transferred to a beaker and the solution was heated to 800C. NaOH (0.1 N) was added drop wise till the pH reached 7.0. The broth was filtered using a filter paper and the filtrate was discarded. The precipitate collected from filter paper was dissolved in a conical flask using minimal volume of 0.1 N HCl. After dissolution of the precipitate, the solution was titrated against 0.1 N NaOH using phenolphthalein as indicator. The titratable acidity was calculated as lactic acid % w/v (Fortina et al., 1973). Each millilitre of 1 N NaOH is equivalent to 90.08 mg of lactic acid. The titratable acidity was then calculated (2).
where X is the total volume of NaOH
Y is the volume of NaOH consumed during the titration of 5 ml broth.
Z is the total amount of broth.
Results and discussion Characterization of Dairy effluent sample:
The various physiochemical parameters such as pH, COD, BOD, TDS etc of the effluent were estimated and given in Table 1. The current effluent analysis indicated elevated concentration of BOD and COD as 3200 and 2280, respectively (where the permissible range of BOD and COD are 40 mg/l, 120 mg/l, respectively and The Total Dissolved solids (TDS) and Total suspended solids (TSS) were found 1744 to 453, respectively (Table 1). The pH of the sample was determined as 7.3 (Table 1).
Table 1. Characteristics of the wastewater collected from dairy plant.
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Total Dissolved solids (TDS) (mg/l)
Total Suspended solids (SS) (mg/l)
Screening of microorganisms for lactic acid production: Four bacterial populations with distinct colony morphology and colour were identified and isolated in pure form from isolation plates and subjected to screening for LA production in Agar media containing 25-200 mg/l of dextrose. The majority of the isolates were able to grow up at 50 mg/l dextrose concentration but their number decreased as the dextrose concentration in the media was increased. All other strains except Lactobacillus casei showed no visible growth above 200 mg/l dextrose concentration. In this way Lactobacillus casei was isolated from dairy effluent.
Biochemical & morphological characterization of Lactobacillus casei: The morphological, biochemical & carbohydrate utilization analysis confirmed that the isolate was a member of the genus Lactobacillus and formed a subline within the Lactobacillus casei cluster (Table 2).
Table 2. Morphological and biochemical characteristic of Lactobacillus casei.
Optimized culture conditions for lactic acid production: From the experimental results it was evident that the suitable temperature and pH for lactic acid production was found to be 280C and 6.5, respectively (Fig. 1 & 2). The effect of incubation temperature on lactic acid production was studied by incubating the culture Ã¯Â¬â€šasks at various temperatures like 26, 28, 30, 32, 340 C. Fig. 1 showed that at 280C, maximum lactic acid was produced. The culture showed good lactic acid production also at 300 C temperature.
Fig. 1. Temperature optimization for Lactic acid production. Every individual data was taken after 20 hrs of culture growth.
It was found that pH 6.5 produced maximum concentration of lactic acid in the broth by L. Casei. It was also found that in the range of pH 5.5 -8.5 the lactic acid production was quite stable. Lactic acid production by the bacterial cells was drastically reduced beyond the pH 9.0. Since after 20 hr culture there was negligible change in the extra cellular concentration of lactic acid, all samples were taken after 20 hr of growth.
Fig. 2. pH optimization for lactic acid production. Data taken after 20 hrs of growth.
Various carbon sources like sucrose, starch and dextrose were administered in the culture flask to investigate the effect of lactic acid production by L. Casei. Peptone was used as the only nitrogen source. The highest lactic acid activity of the strain was obtained when dextrose was used as the carbon source in the media (Fig. 3).
Fig 3- Medium optimization for LA production. Data taken after 20 hrs of growth.
It was also essential to determine the volume of inoculums or inoculums size (the volume of seed bacterial culture to the volume of new culture medium) for the bacterial growth. The study was conducted using different size/volume of inoculums like 0.5%,1%,1.5%,2%,3%,4%,5% using pH 6.5, temperature 300C and shaking speed of 250 rpm. After 12 hours and 20 hours of incubation maximum lactic acid production was observed with 1 % inoculum volume while lowest level of lactic acid production was observed with 5% inoculum volume (see Fig. 4).
Fig. 4. Plot of inoculums size vs. for lactic acid production. Various inoculums size was used to see the production level of lactic acid after 2 hr & 24 hr of bacterial culture. The lactic acid activity assay was carried out in cell free medium & plotted against inoculums size.
Fig 3- Kinetics of Strach hydrolysis and Lactic acid production at pH -7 and Temperatur-280C
Conclusion: In conclusion, various LAB do exist in the dairy effluent samples that were collected from OMFED dairy plant of Rourkela. For the economical considerations, lactic acid fermentations from dairy resources were investigated. These studies clearly indicated that the dairy waste can be utilized for lactic acid production at its lower concentration effectively using the identified species Lactobacillus casei.