Lactic acid is an organic acid that is important to industry and biology. It has been widely used in foods, chemicals and pharmaceuticals and is increasingly important in the production of biodegradable polymers such as polylactic acid. This biological process is preferred as it yields highly purified lactic acid and has less environmental effect than the traditional chemical process, due to the global movement to more environmentally friendly and sustainable technologies.

Whey is one of the important by-products of dairy and cheese industries. High in lactose, soluble proteins and minerals. It is, however, highly polluting the environment because of its high organic content when not disposed of properly. A major step toward lowering environmental pollution and attaining economic sustainability in the food industry is the use of whey in bio-ferments [3].

Lactobacillus acidophilus is a homofermentative lactic acid bacterium (LAB) which has high potential to produce lactic acid from sugars, in particular from lactose. This bacterium is a very good strain for lactic acid production, as it is able to grow on lactose rich medium such as whey and is somewhat tolerant to acid. In many studies, it has been proven that supplementing L. acidophilus to whey fermentation can markedly enhance the concentration of lactic acid, particularly when the conditions of the fermentation process are optimal [4,5].

Bifidobacterium bifidum is an anaerobic (or semi-anaerobic) bacterium that produces lactic acid and acetic acid in different proportions by means of a special metabolic pathway called the fructose-6-phosphate pathway. Bifidobacterium is remarkable in that it can make use of lactose and improve the final product's functional qualities, although its production of lactic acid is less than for some other species of the genus Lactobacillus. Recent studies have shown that Bifidobacterium species can generate more lactic acid under the appropriate growth conditions and growth factors [6,7].

It has been reported that whey could be used more efficiently when the following catalysts are added: organic nitrogen source, mineral salts, vitamins and lactases. These catalysts activate lactic acid bacteria, facilitate the fermentation process and increase yield. The temperature,pH and the concentration of the substrate also need to be controlled, in order to produce the process that is efficient. This emphasizes that combining the choice of the right strain with improving fermentation conditions is a good way to use whey as a cheap and long-lasting source of lactic acid [8].

A study will be conducted to evaluate the possibility of investing in whey as sustainable carbon source for lactic acid production by using the bacteria Lactobacillus acidophilus and Bifidobacterium bifidum; aiming to maximize the benefits of the dairy waste and at the same time to minimize its environmental impacts in the frame of sustainable development. Also, it will study the effect of some of the aid factors on improving the fermentation efficiency and lactic acid productivity.

Single cultures of Lactobacillus acidophilus and Bifidobacterium bifidum were obtained and activated in vitro at the Food Science Laboratories of the University of Mosul.

Whey was obtained as a by-product of the enzymatic production of white cheese made from buffalo milk using microbial rennet. The whey was then heated at 90°C for 20 minutes to precipitate the proteins and the precipitated proteins were then removed by centrifugation at 4000 × g for 15 minutes [9].

Lactic Acid Determination

The approach outlined was followed with some modifications. This technique uses the optical absorption of the greenish yellow ferric lactate compound formed when lactate reacts with ferric chloride (FeCl3) at a wavelength of 405 nm with an optical spectrometer made by APEL Japan. The calculation of lactic acid concentration was done by the sheet [8] equation and the law:

Using the meal method and a mixed culture of B. bifidum and L. acidophilus incubated at 40°C, the impact of various factors on the production of lactic acid from whey was investigated. The vaccine of isolates was prepared using the method of Chavarri [10]. According to the methods described by Sheet et [8], this included the type and concentration of carbon and nitrogen sources, some mineral elements, the concentration and age of the sample, the effect of acidity function and the length of the incubation period.

Type of Nitrogen Source's Impact on Lactic Acid Production

The whey medium used to produce acid was supplemented with a few nitrogen sources at a concentration of 10 g/L. Ammonium nitrate, sodium nitrate, ammonium chloride, meat extract, urea, peptone and yeast extract were among the additional sources. Each lactic acid was estimated following fermentation. Regarding how the concentration of yeast extract affected the production of lactic acid, concentrations ranging from 2 to 20 g/L were employed. The fermentation process.

Type of Carbon Source's Impact on the Production of Lactic Acid

At a concentration of 10 g/L, sugars were added to the whey medium that had the best amount of yeast extract as a source of carbon. The fermentation and acid production process was done and after the fermentation was done, the amount of lactic acid was measured.

Magnesium Sulfate Concentration's Impact on the Synthesis of Lactic Acid

The whey medium with the right amount of yeast extract was mixed with lactose sugar at levels between 5 and 50 g/L. Then, a process was done and the amount of lactic acid made was measured.

Manganese Sulfate Concentration's Impact on the Synthesis of lactic Acid

We added manganese sulfate to the whey medium that already had the right amounts of yeast extract and lactose. The amounts of manganese sulfate ranged from 0.01 to 0.1 g/L. The fermentation process was done with a mixture of bacteria and lactic acid that was guessed.

Impact of Vaccine Concentration on the Production of Lactic Acid

The bacterial vaccine, at concentrations ranging from 5 to 20 × 10⁷ cells/ml, was added to whey medium fortified with optimal concentrations of yeast extract, lactose, magnesium sulfate and manganese sulfate. Fermentation was then carried out and lactic acid was measured.

Vaccine Age Effect

Bacterial vaccine was prepared by inoculating sterile liquid culture media with the Acido and Bifido strains and leaving them to incubate for different periods (20, 24, 48 and 72 hours) at 37°C to produce different physiological ages of bacterial cultures. The 20- and 24-hour incubations are the active logarithmic growth phase and the 48- and 72-hour incubations are the static and dormant phase. The cell count of all age classes were standardized with spectroscopy after each incubation period to make valid comparisons when used as a starter culture during lactic acid production.

Impact of Incubation Duration on the Production of Lactic Acid

The incubation of the whey medium with the bacterial mixture took place over a period of 6 to 48 hours. At the end of the incubation periods, the amount of lactic acid made was measured.

pH Impact on the Production of Lactic Acid

We made the whey medium by adding the ingredients listed above. Then, we used hydrochloric acid or sodium hydroxide (1 molar) to change the pH to between 4 and 8. After that, we sterilized the medium, added the vaccine and started the fermentation process. Lactic acid was measured after the fermentation period was over.

The Difference in Productivity with or Without Factors

The lactic acid production of both strains in whey medium was compared under two treatments: the first without adjuvants (control) and the second with the addition of optimal factors (carbon and nitrogen sources, mineral elements and control of pH, age and vaccine ratio). The cultures were incubated under the appropriate conditions and the lactic acid concentration was then estimated.

Statistical Analysis

To determine the type of differences between treatment levels, a straightforward experiment with a completely randomized design (CRD) was used to analyze the data. At a probability level of 0.01, the significance of mean differences was assessed using Duncan's multiple range test. Three replicates of each treatment were used in the statistical analysis, which was carried out using the SAS statistical software.

The Effect of Different Types of Protein Sources

The results shown in Figure 1 indicate that five materials tested had an effect on lactic acid production with the Acido and Bifido strains. In the case of Acido, productivity reduced significantly with the use of urea and ammonium chloride and was the lowest when ammonium sulfate 1.25 g/L was used. The productivity of the Bifido strain was generally lower than that of the Acido strain and was highest on yeast extract 1.25 g/L and lowest on ammonium sulfate 0.25 g/L. The substantial variations in these values suggest that the protein source had a marked influence on the efficiency of lactic acid production with both strains. These results can be attributed to the presence of free amino acids, short peptides and growth factors in complex protein sources like yeast extract and peptone that is essential for improving the metabolic activity of bacteria and subsequently optimizing the fermentation processes, thereby a higher amount of lactic acid is produced [11].

Figure 1: The Impact of Incorporating Nitrogen Sources on Lactic Acid Synthesis

Bifido: B. bifidum, Acido: L. acidophilus

*Three replicates are averaged to determine the values. * Significant differences at the 0.01 level are indicated by letters above columns

Effect of Sugar Source Types

In Figure 2 Effect of different sources of sugar shows a great variation in production of lactic acid for both strains under different treatments. The glucose 35.50 g/L resulted in the highest production followed by lactic acid 33.94 g/L, while production was significantly decreased with sucrose 30.85 g/L and fructose 30.11 g/L. The Bifido strain showed the highest value with the use of lactose (15.25 g/L) followed by glucose (13.95 g/L) and the lowest values were obtained with the use of sucrose (12.44 g/L) and fructose (12.05 g/L). The results can be attributed to the fact that glucose and lactose are two of the most metabolically efficient sugars for lactic acid bacteria. The glucose is directly added to the glycolysis pathway to increase production of pyruvate and lactic acid, while lactose is converted in the cell into glucose and galactose which increases metabolic activity especially in the whey medium, which is high in nutrients [12]. Other sugars, on the other hand, need other enzymes or less efficient transport mechanisms, which have a negative effect on the fermentation rate and the acid production.

Figure 2: The Impact of Incorporating Carbon Sources on Lactic Acid Synthesis

Bifido: B. bifidum, Acido: L. acidophilus

* Three replicates are averaged to determine the values. * Significant differences at the 0.01 level are indicated by letters above columns

Effect of Multiple Concentrations of Magnesium Sulfate

As seen in Figure 3, the production of lactic acid was significantly affected by the addition of magnesium sulfate.The Acido strain produced the highest yields at concentrations 0.2, 0.3 and 0.35, where the yields ranged from 31.91 to 32.05 g/L and were not significantly different. The Bifido strain, in contrast, obtained the highest production value at 0.1 and the production value started to drop with the increasing concentrations above 0.1. The explanation of the above results is as follows: Magnesium ions are essential coenzymes for many enzymes which are concerned with fermentation processes. But a higher concentration can result in an inhibitory effect or to an imbalance of the intracellular ionic balance [13].

Figure 3: The Influence of Manganese Sulfate Addition on Lactic Acid Synthesis

Bifido: B. bifidum, Acido: L. acidophilus

* Three replicates are averaged to determine the values. * Significant differences at the 0.01 level are indicated by letters above columns

Effect of Multiple Concentrations of Manganese Sulfate

The effect of manganese sulfate on the lactic acid production is shown in Figure 4. The Acido strain had the maximum productivity at 0.07, with a productivity of 39.87 g/L, which decreased with increasing concentration. The Bifido strain grew best at 0.07 and 0.2 and there was no significant difference between the two. This is thought to be due to manganese's ability to activate certain enzymes which are important for reducing oxidative stress and cell growth. But any levels above the optimum can cause inhibitory effects to metabolic activity [14].

Figure 4: The Impact of Incubation Duration on Lactic Acid Synthesis

Bifido: B. bifidum, Acido: L. acidophilus

* Three replicates are averaged to determine the values. * Significant differences at the 0.01 level are indicated by letters above columns

Effect of Incubation Duration

Figure 5 illustrates the effect of incubation duration on the metabolic activity of the Acido and Bifido strains. The Acido strain recorded its highest productivity at 24 hours, reaching 35.03 g/L, followed by 30 hours at 34.55 g/L. Productivity then gradually declined over time. 

The Bifido strain reached its peak activity at 30 hours with a productivity of 16.32 g/L, with a noticeable decrease observed at other time periods. Overall, the Acido strain was observed to be superior in all studied incubation periods. These results can be interpreted as the peak at 24-30 hours representing the highest stage of metabolic activity for the strains, while the subsequent decline is attributed to nutrient depletion in the medium or a decrease in pH due to acid accumulation, leading to growth inhibition [15].

Figure 5: pH Impact on Lactic Acid Synthesis

Bifido: B. bifidum, Acido: L. acidophilus

* Three replicates are averaged to determine the values. * Significant differences at the 0.01 level are indicated by letters above columns

pH Impact

As is evident from Figure 6, the production of lactic acid by the two strains was greatly affected by pH with a maximum of 26.30 and 35.33 at pH 6 for the two strains respectively followed by maximum values at pH 6.6 and 7. Yield decreased at lower acidic pH values such as pH 4 and 4.5 (26.30 and 27.00) and also at higher alkaline pH values such as pH 7.5 and 8 (32.17 and 31.50). Bifido showed lower yields ranging from 12.90 to 16.20, with the highest yield recorded at pH 6.6 (16.20), followed by pH 6 and 7 (15.30 and 14.99). Yield was reduced at high acidic and alkaline pH. The results have shown that any values close to the neutral range will be the most suitable for the activity of enzymes responsible for fermentation of sugars and production of lactic acid, whereas any point away from the neutral range will be in favor of the decrease in metabolic activity and cell growth [16].

Figure 6: Lactic Acid Production Variation with and Without Factors

Bifido: B. bifidum, Acido: L. acidophilus

* Three replicates are averaged to determine the values. * Significant differences at the 0.01 level are indicated by letters above columns.

The Difference in Productivity with and Without Factors

The productivity difference with and without cofactors is shown in Figure 6. Under optimal conditions and factors, a marked rise in the lactic acid production was noticed for both the strains. The production of the Acido strain was higher with cofactors (58.26 g/L) than without (38.45 g/L). Likewise, under the same conditions, the production of the Bifido strain increased from 30.10 g/L to 45.50 g/L, which is an indication of the complementary positive effect of these factors in increasing the efficiency of the fermentation process. All these factors contribute to increasing the efficiency of the enzyme systems which convert sugars to lactic acid, decrease metabolic stress on cells and increase cell membrane stability and nutrient uptake. In a study by Sheet [8], it was shown that lactic acid bacteria can be used in enriched whey medium to increase acid production, which was highlighted in this study.

The Acido strain showed more aptitude to make use of optimal conditions than the Bifido strain. This is related to different genetic composition and efficiency of the metabolic pathway [17,18]. These results echoed that the improvement of the fermentation factors not only boosted the productivity but also is able to maximize the process efficiency and minimize the process loss, thereby making the use of enriched whey medium in bioproduction more feasible.

The study showed that whey is a promising biological substrate for lactic acid production, giving one of the tools for sustainable development through recycling of dairy by-products. The results showed that there were significant differences in the genetic and metabolic characteristics between both strains, L. acidophilus having higher nutrient utilization efficiency and greater productivity than B. bifidum. Maximizing the bioavailability of these complex carbon (lactose) and nitrogen sources (yeast extract) as well as the control of mineral elements and physical conditions (pH and vaccine age) is critical to the integration. Optimal use of fermentation conditions by Acido strain resulted in an increase in productivity of more than 50%, which proves the environmental conditioning of the fermentation medium to reduce the metabolic stress and favor the enzyme systems, thus highly economically viable.

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