Lactic acid is one of the most widely used carboxylic acid. It is utilized in food industry, pharmaceutical, cosmetic, bioplastic industry, etc. Lactic acid is an intermediate chemical to produce propylene glycol, propanoic acid, acetaldehyde, ethyl lactate, lactic acid polymers, and aniline. Global market volume of lactic acid in 2021 reached 1.39 million metric tons, which is 48% higher than its volume in 2016. The market demand of lactic acid is predicted to reach 2.65 million metric tons in 2029 (Statista 2023).

Lactic acid is commercially produced by reaction of acetaldehyde and hydrogen cyanide in high pressure, proceeded with hydrolysis of lactonitrile. This process requires intensive safety procedure considering highly poisonous properties of hydrogen cyanide. Another commercial production route of lactic acid is fermentation of milk carbohydrate using enzyme complex of Lactobacillus sp, which is also applied around the world.

Biochemically, lactic acid is produced in plants and animals from pyruvic acid using lactate dehydrogenase enzyme. This reaction is a part of biosynthetic pathways producing propionic acid, lactic acid, and acetic acid from glycerol (Liu et al. 2016). It shows a possibility of lactic acid production from glycerol using enzyme complex. Lactic acid production from glycerol has been an interesting research subject in the last decade, considering high availability of glycerol from oleochemical and biodiesel industry. Conversion routes of glycerol to lactic acid using metal catalysts have been investigated, but most process requires robust process condition (Arcanjo et al. 2017; Purushothaman et al. 2014; Ramírez-López et al. 2010; Razali et al. 2017). Utilization of enzyme complex involving lactate dehydrogenase offers alternative route with milder condition, mimicking biosynthesis of lactic acid in organisms.

Lactate dehydrogenase (LDH) is present in rice (Oryza sativa) and corn (Zea mays). LDH was extracted successfully from rice (Rivoal et al. 1990) and corn (Christopher et al. 1996) with low temperature method, which is applicable and economically feasible. The activity of LDH crude extract from rice and corn were reported (Christopher et al. 1996; Rivoal et al. 1990) although the optimum temperature of extraction and pyruvic acid conversion have never been reported. These successful experiments of LDH extraction emerges a potential to convert pyruvic acid to lactic acid in moderate temperature, considering LDH and most enzymes active in temperature below 100℃. Pyruvic acid conversion to lactic acid is a subsequent step in glycerol metabolism route, thus development of enzymatic pyruvic reduction is an important step to establish conversion of glycerol to lactic acid technology.

In this research, lactate dehydrogenase activity of rice extract and corn extract for conversion of pyruvic acid to lactic acid was investigated. Effect of extraction temperature, reaction temperature, and extract ratio to substrate into lactic acid concentration were also determined.

Extraction of lactate dehydrogenase

Extraction of LDH from rice and corn extract was performed with procedure from Rivoal et al. (1990). Rice seeds and corn seeds were grown for 7 days in vermiculite. Grown seeds and roots were grinded and homogenized in room temperature with solvent mixture (200 μL for 5 seedlings or 2 mL/g root tissue) which consist of 20 mM Tris-HCl (Sigma Aldrich, >99%, pH of 8), 5 mM Na-ascorbate (NOW Foods, pharmaceutical grade), 1 mM EDTA (Himedia GRM678 >98%), and 10%-v/v glycerine (Sigma Aldrich, 99%). Homogenized mixture was incubated for 20 hours at specified temperature (5℃, 27℃, and 37℃), then centrifuged for 15 minutes at 12000g. Supernatant was collected and an aliquot was analysed with Bradford protein assay (Bradford 1976).

Reaction using LDH crude extract

Crude LDH from rice or corn extract was added into pyruvic acid with specified ratio (5%, 10%, 15%, 20%, and 30%-v/v). Pure nitrogen gas was flowed into reaction to minimize oxidation. Reaction was performed for 5 hours at specified temperature (27℃, 37℃, 47℃, and 57℃). Reaction product was diluted in H₂SO₄ 0.005 M and analysed with High Performance Liquid Chromatography (HPLC) Rezex TM ROA-Organic Acid H+ 8% (300 x 7.8 mm column) (Artini et al. 2018). Lactic acid yield production yield was quantified from chromatograph peaks. Lactic acid analytical standard (Sigma Aldrich) was used as reference.

Effect of extraction temperature

Enzyme concentration in rice and corn extract was determined with Bradford protein assay. The effect of extraction temperature to protein concentration is illustrated in Fig. 1. Rice extraction yields in higher protein concentration than corn extraction. There is no correlation shown between extraction temperature and protein yields, thus there is no necessity in increasing solvent temperature for higher protein solubility.

Figure 1. Effect of extraction temperature to protein concentration.

Effect of reaction temperature

The effect of reaction temperature to lactic acid production with lactate dehydrogenase (LDH) is illustrated in Fig. 2. Although protein concentration in rice extract is significantly higher than corn extract (Fig. 1), their activity on pyruvic acid reduction is similar. It indicates higher content of non-LDH enzyme in rice extract than in corn extract, which do not contribute to pyruvic acid reduction.

Figure 2. Effect of reaction temperature to lactic acid production with lactate dehydrogenase.

Activity of pyruvic acid reduction into lactic acid was optimum at 47℃ for corn extract and 57℃ for rice extract. These temperatures are relatively moderate compared to LDH from other sources (including alga, bacteria, and animal), as illustrated in Fig. 3.

Figure 3. Optimum temperature of lactate hydrogenase from various sources: Oryza sativa and Zea mays (this research), Dunaliella salina, Chlamydomonas reinhardtii, porcine muscle (Saburova et al. 2000), Lactobacillus delbrueckii (Sun et al. 2021), Litopenaeus vannamei (Leyva-Carrillo et al. 2019) and Thermus caldophilus (Taguchi et al. 1982).

For enzyme applications, mild reaction temperature is preferred to minimize heating or cooling, making the process more economic. High temperature also tends to denature enzymes (LDH and others) present in reaction mixture. Fig. 2 shows that LDH from corn extract facilitates similar activity compared to LDH from rice extract at milder temperature, which is an advantage for further application.

Effect of LDH crude extract concentration

The effect of rice and corn extract concentration to pyruvic acid reduction (quantified by determination of lactic acid production) is illustrated in Fig. 4. Both rice and corn extract shows zero activity when only 5% extract was involved and increased activity on 10% extract, and relatively stagnant above 20% extract. Higher concentration of enzyme promotes more contact of substrates, but also causes more inhibitions. In the case of crude enzyme utilization, side reaction are likely to occur, limiting production of main reaction products. The analysis of side reactions are described in the following section.

Figure 4. Effect of extract concentration to lactic acid production with lactate dehydrogenase.

Overall, both Figs. 3 and 4 shows that rice and corn extracts obtained with Rivoal et al. (1990) procedure were able to perform activity of pyruvic acid reduction to produce lactic acid. It also confirms the feasibility of using water in reaction mixture as source of H⁺ in pyruvic acid reduction. Though crude extract from rice and corn are practically easy to use, it should be noted that the enzyme content is less consistent compared to properly isolated enzyme.

Experiments in previous sections, though confirmed the activity of lactate dehydrogenase in rice and corn extract, shows the limitation of lactic acid production which is not caused from temperature or enzyme content. Quantitative analysis of lactic acid using HPLC, both in rice and corn extract (Figs. 5 and 6), shows existence of other substances in reaction products. Lactic acid was shown at retention time 14.0-14.1 minutes, while other products were shown at 11.3-11.7 minutes (will be mentioned as X-product), and at 14.6 minutes (will be mentioned as Y-product). Considering peaks total areas in HPLC result are proportional to their respective concentration, it can be concluded that X-product and Y-product concentration was much higher than the lactic acid produced.

Figure 5. Product quantification of experiment with rice extract (temperature 47℃, 20% extract, 5 hours, with agitation).

Figure 6. Product quantification of experiment with corn extract (temperature at 47℃, 20% extract, 5 hours, with agitation).

To confirm the origins of X- and Y- products, another experiments was conducted under the same condition, only excluded the LDH extracts. Pyruvic acid was stirred in similar manner to previous experiment (with nitrogen blanketing, for 5 hours at 47℃), without addition of rice or corn extracts. Under the absence of crude LDH, it is clear that Y-product was not formed, while the X-product was still present (Fig. 7). It suggest that Y-product formation was catalysed by enzyme complex in rice and corn extracts, while X-product was not. Lactic acid was also not present in this product, confirming the crude LDH role to produce lactic acid from pyruvic acid.

Figure 7. Product quantification of experiment without rice or corn extract (pyruvic acid heating at 47℃, 5 hours, with agitation).

Given that reaction was conducted in oxygen-minimized (not completely inert) condition, X-product was predicted as lactic acid oxidation product, considering several spontaneous reactions can be formed by oxidation of pyruvic acid (Table 1). Thus, X-product occurred in the reaction was predicted to be malonic acid, or hydroxypyruvic acid, tartronic acid. To develop lactic acid production using crude LDH extract from rice and corn, it is recommended to investigate the effect of oxygen presence in process (for example, with adjusted nitrogen flow).

Table 1 . Potential products of pyruvic acid oxidation.

Substance	Reaction	Gibbs free energy (ΔGo)
Malonic acid	C3H4O3 + ½O2 → C3H4O4	-136.82
Hydroxypyruvic acid	C3H4O3 + ½O2 → C3H4O4	-93.72
Tartronic acid	C3H4O3 + O2 → C3H4O5	-276.08

While X-product was formed with the absence of LDH extract, Y-product occurs only when rice or corn extract was involved. It indicates that Y-product was caused by side reaction from crude extracts. It is confirmed by growing peak of Y-product with increasing amount of LDH extract, as shown in Fig. 8.

Figure 8. Product quantification of experiment at temperature 26℃, 5 hours, with rice extract (a) 5% extract; (b) 10% extract; (c) 20% extract; (d) 30% extract; (e) 40% extract.

There are many possible enzyme exist in rice and corn extract, considering the complex reactions in pyruvic acid metabolic pathways (Maleki et al. 2017). Bioconversion reactions of pyruvic acid is listed in Table 2.

Table 2 . Bioconversions of pyruvic acid in organism (Maleki et al. 2017).

Product	Promoting enzyme	Reaction
Phosphoenol pyruvate (PEP)	PEP synthase	Pyruvate + ATP + H2O → PEP + AMP + Pi
Acetaldehyde	Pyruvate decarboxylase	Pyruvate → CO2 + acetaldehyde
Acetate	Pyruvate oxydase	Pyruvate + ubiquinone → acetate + CO2 + ubiquinol
Oxaloacetate	Pyruvate carboxylase	Pyruvate + HCO3- + ATP → oxaloacetate + ADP
Acetyl CoA	Pyruvate format lyase	Pyruvate + CoA → Acetyl CoA + formate

Experiment procedure in this research did not provide ubiquinone, HCO₃^-, or CoA in considerable amount, thus only PEP and acetaldehyde synthesis was feasible to occur under the condition. Pyruvate decarboxylase, the enzyme contributing to acetaldehyde biosynthesis, was reportedly active in several plant extracts, including rice extract (Hossain et al. 1996; Jain et al. 2020; Li et al. 2004; Mohanty et al. 2003; Rivoal et al. 1990) and corn extract (Christopher et al. 1996; Lee et al. 1985) suggesting that acetaldehyde is possibly main product formed in reaction with LDH extract. There has not been any report of phosphoenolpyruvate (PEP) synthase activity in rice or corn extract, though further identification should be performed to confirm the substance.

Lactate dehydrogenase from rice (Oryza sativa) and corn (Zea mays) extract was obtained and their activity of pyruvic acid reduction to lactic acid was confirmed. There was no correlation shown between extraction temperature and protein yields, while the optimum temperature for pyruvic acid reaction to lactic acid was 57℃ (with LDH from rice extract) and 47℃ (with LDH from corn extract).

Under presence of oxygen, reaction selectivity tends to promote pyruvic acid oxidation (possibly into malonic acid, or hydroxypyruvic acid, tartronic acid). Increasing amount of enzyme contribute to production of more side product instead of lactic acid. This side product was predicted to be acetaldehyde, considering the presence of pyruvate decarboxylase enzyme which was also found in rice and corn extract.

Lactate dehydrogenase from rice and corn extract was confirmed active, whether it will be developed to produce lactic acid or other derivatives from pyruvic acid. It is necessary to proceed investigation of reaction selectivity and products identification. This reaction will be one of sequential reactions involved that can be utilized for glycerol conversion.

The author would like to thank Fakultas Teknologi Industri Institut Teknologi Bandung for their support through Penelitian, Pengabdian Masyarakat dan Inovasi program.