The Kingdom of Saudi Arabia produces over one million metric tons annually, which returns a 20% of wasted dates annually. Lactic acid and its derivatives are widely used in food, pharmaceutical and textile industries. There has been an increase in lactic acid production because it is used as a raw material to produce polylactic acid, a polymer that is used as a special medical and environmental friendly biodegradable plastic. This study aimed to use wasted dates to produce lactic acid by single culture Lactobacillus casei (ATCC 393), Lactobacillus acidophilus (CICC 6088) and the mixed culture using batch fermentation. The investigation results showed that the maximum concentration of lactic acid for ATCC 393, CICC 6088 and the mixed culture are 87, 84 and 84 g/l respectively. For single CICC 6088 and the mixed culture, the total percentage of glucose and fructose utilized was found to be 100%; 76%, respectively, whereas in the case of the single culture ATCC 393, the total percentage of glucose and fructose were 100% and 72%, respectively. With regard to lactic acid concentration, and sugar consumption, the results revealed that the single culture ATCC 393 produced the optimum lactic acid of 87 g/l for 48 hr with initial sugar concentration of 90 g/l.
The annual production of palm dates in the Kingdom of Saudi Arabia (KSA) is estimated to be over 1 million metric tons in 2013 [
The strains of Lactobacillus casei (ATCC 393) and Lactobacillus acidophilus (CICC 6088) are homofermentative bacteria that produce lactic acid. These strains were kindly purchased from (ATCC company, USA) and (CICC company, China) and stored as freeze-dried substances. The ATCC 393 and CICC 6088 were prepared from ATCC medium and CICC mediumas shown in
Medium for ATCC 393 | Medium for CICC 6088 |
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10 g/l Peptone 10 g/l Beef extract 5 g/l Yeast extract 5 g/l Sodium Acetate 2 g/l Na2HPO4 0.1 g/l MgSO4.7・H2O 0.05 g/l MnSO4・H2O 1 g/l Tween-80 20 g/l Dextrose | 10 g/l Peptone 10 g/l Beef extract 5 g/l Yeast extract 5 g/l Sodium Acetate 2 g/l K2HPO4 2 g/l MgSO4.7・H2O 0.05 g/l MnSO4・H2O 1 g/l Tween-80 20 g/l Dextrose |
The inoculum was prepared by transferring 5 ml of culture to a 250 ml Erlenmeyer flask containing 100 ml of liquid (ATCC and CICC) Medium for pre-culture. The flask was subsequently incubated at 37˚C and 200 rpm for 24 hr.
The substrate used was obtained from wasted dates. The wasted dates or sorting date discards are a part of the palm tree fruit, which were assessed invalid for human consumption because it has been stored for solong time. The wasted dates were supplied from a date factory belongs to the college of food and agriculture sciences at King Saud University, Riyadh. The dates were thoroughly cleaned manually from dust and other substances. Date fruit is composed of the fleshy part and the seed. The seeds were separated by manual splitting, then two liters of distilled water were added to one kilogram of dates (V/W = 2/1). Then, the mixture was heated at 80˚C for 2 hr with continuous stirring. The resultant dates syrup was centrifuged at 3000 rpm for 15 min to separate the cellulosic debris. After filtration, the dates juice was observed to have mainly 60% of fructose and 40% of glucose, with accordance to High-Performance Liquid Chromatography (HPLC) analysis. With temperature adjusted at 40˚C was diluted with 1 ml H2SO4 as a mobile phase at a flow rate of 0.8 ml/min.
The medium consisted of dates syrup as a substrate of 90 g/l of total sugar (36 g/l of glucose and 54 g/l of fructose) used as the substrate. The medium was sterilized at 121˚C for 15 minutes. After cooling, the medium was supplemented with salt solution containing (0.2 g/l of MgSO4, 0.03 g/l of MnSO4, 0.3 g/l of K2HPO4, 0.3 g/l of KH2PO4, 0.02 g/l of FeSO4 and 1 ml/l tween 80), yeast extracted as a nitrogen source to supplement about 20 g/l.
The batch fermentation cultivations were performed in 250 ml-Erlenmeyer flasks with a working volume of 100 ml. The pH value of the cultures was maintained manually between pH 5 to 7 by adding a 5 N of NH4OH solution each 6 hours, and a fixed temperature of shaker was maintained at 37˚C with an agitation speed of 200 rpm. Flasks containing the production medium of sugar concentration were inoculated with a portion of the microorganism culture of Lactobacullus casei (ATCC 393), Lactobacullus acidophilus (CICC 6088) and mixed culture of both. The mixed culture was obtained as a 50% of (ATCC 393) and 50% of (CICC 6088). Aninoculum (with a percent of 20%) grown in the medium of lactobacillus (ATCC 393, CICC 6088 and mixed culture) was used in all fermentation processes.
The cells concentration was estimated by measuring the optical density at a wavelength of 620 nm. Lactic acid, glucose and fructose concentrations were determined by High-Performance Liquid Chromatography system (HPLC Agilent model 1260 infinity, USA, equipped with RI, UV detector at 210 nm), in which an injection volume of sample was set at 5 μl. Analytical guard column (4 × 80 mm) with temperature adjusted at 40˚C was diluted with 1 ml H2SO4 as a mobile phase at a flow rate of 0.8 ml/min.
The cells growth during fermentation for the strains ATCC 393, CICC 6088 and the mixed culture are shows in
In this part, a comparison was conducted between sugar utilization and lactic
acid production with single and mixed culture during fermentation. The utilization of glucose and fructose from dates juice has been achieved by a single ATCC 393, CICC 6088 and a mixed culture of these strains. The results are shown in
76% in fermentation with ATCC 393, CICC 6088 and the mixed culture, respectively. It was noted that in sugar utilization, the glucose was consumed early by the three cultures when comparing with fructose. This reveals that the selected bacteria strains (ATCC 393, CICC 6088 and mixed culture) prefer glucose than fructose based on our specific conditions that prepared for fermentation processes.
Production of Lactic acid (as shown in
Kinetic Parameters (48 hr) | ATCC 393 | CICC 6088 | Mixed Culture |
---|---|---|---|
Maximal Lactic acid concentration, (g/l) | 87 | 84 | 84 |
Maximal Lactic acid productivity, (g/l・hr) | 1.81 | 1.75 | 1.75 |
Maximum Cells growth (OD) | 1.75 | 1.83 | 1.8 |
Percent glucose utilized (%) | 100 | 100 | 100 |
Percent fructose utilized (%) | 72 | 76 | 76 |
The study was intended to use wasted dates to produce lactic acid by single culture Lactobacillus casei (ATCC 393), Lactobacillus acidophilus (CICC 6088) and the mixed culture through batch fermentation. The investigation results showed that the highest concentration of lactic acid was 87 g/l archived by ATCC 393, While CICC 6088 and the mixed culture produced 84 g/l as their highest concentrations. The summarized conclusion of the study is that the single culture ATCC 393 has achieved the optimum lactic acid concentration (which was 87 g/l) for 48 hrs with initial sugar concentration of 90 g/l with regards to lactic acid concentration and sugar consumption. Hence, upon the study findings, it is worthy to note that wasted dates could be an attractive substrate for the lactic acid production by fermentation.
The authors gratefully acknowledge the financial support by Research Center, College of Food and Agriculture Sciences at King Saud University.
Bushara, M.H., Alkoaik, F., Abasaeed, A. and Fulleros, R. (2018) Fermenting Saudi Wasted Dates by Using Lactobacillus casei (ATCC 393), Acidophilus (CICC 6088) and the Mixed-Culture Bacteria to Produce Lactic Acid. Open Journal of Applied Sciences, 8, 150-157. https://doi.org/10.4236/ojapps.2018.84012