Use of Lactobacillus for Lactic Acid Production from Agro-Industrial By-Products

Abstract

Agro-industrial by-products have not been efficiently valorized. Lactobacillus used to transform these by-products into interesting metabolites is a way to increase the adding-value of these residues and to contribute to the circular economy. These lactic acid bacteria (LAB) metabolize the available substrate produced by enzymes that are responsible for breaking complex carbohydrates into glucose and subsequently obtaining lactic acid through glycolysis in a homofermentative process. By-products used like substrates to produce lactic acid must be rich in carbohydrates e.g. whey, cassava peel, pineapple peel, and molasses, among others. In addition, from lactic acid obtained, it is possible to develop functional foods such as easily-assimilated beverages and to be antagonists to pathogenic microorganisms such as E. coli, improve the quality of final products and extract compounds of interest like pigments.

1. Introduction

Agribusiness is a productive activity that combines the industrial and agricultural processes to obtain products with added value, which can have food or non-food applications [1]. As a result of the agro-industrial activity, waste is generated in significant quantities that represent an environmental problem, due to the inefficiency of its use or the lack of knowledge about appropriate methods for its treatment [2]. Agro-industrial waste is defined as a solid or liquid material product of the processing of primary products, which are not used in the production process. However, these can be used to generate another product of greater economic or ecological value [2].

The use of agro-industrial by-products obeys the guidelines of the circular economy, where the concept of waste disappears, and this element will be used as a resource for nature, society, and industry. The circular economy aims to avoid the creation of waste that can have a negative impact on the environment, climate, and health [3]. In addition, the use of agro-industrial by-products may contribute to the Sustainable Development Goals (SDGs) of The United Nations Development Program (UNDP), in particular, “no poverty” and “zero hanger”.

The use of LAB to valorize food by-products through the production of lactic acid, has generated interest in recent years. LAB are microorganisms that are widely used in the food industry since they allow obtaining fermented products with pleasant sensory characteristics. In addition, to favor the intestinal biota, they have been attributed properties such as the ability to remove toxic metals from aqueous solutions [4], generate unfavorable environments for the growth and development of pathogenic microorganisms, such as Escherichia coli, Listeria monocytogenes, Pseudomonas, etc. [5]. Also, LAB as Lacticaseibacillus and Lactobacillus acidophilus strains have a positive in vivo response to reduce the bioavailability of methyl mercury (CH₃Hg) in the human being [6].

LAB have been classified as Gram-positive bacteria. These microorganisms are facultative anaerobes, they do not form spores and have the shape of bacilli. The main metabolite generated for LAB in the fermentation process is lactic acid, an organic acid very interesting in industry [7]. LAB are demanding microorganisms in terms of nutritional requirements, they need certain amino acids and vitamins of the B complex for their development. The most common media used in the fermentation of LAB include, sugar (glucose, lactose, or sucrose), calcium carbonate (used as a buffer for the medium), and malt germ (for the contribution of nitrogenous elements and growth factors). The optimal fermentation temperature for Lactobacillus is between 15 and 55° C [8] and the fermentation time should be around 6 days, to achieve around a yield of 90% in lactic acid [9].

Lactic acid production may result from homolactic or heterolactic metabolic routes. The homolactic route consists of the conversion of glucose to pyruvic acid, which through the presence of lactate dehydrogenase, acts as an electron acceptor in the oxidation of NADH, becomes lactic acid. Heterolactic fermentation is carried out through the pentose pathway where there is the formation of xylulose-5-phosphate, an intermediate product in the formation of lactic acid [10]. Finally, other products, in addition to lactic acid, are formed such as ethanol, acetic acid, and carbon dioxide [9].

Lactic acid is composed of two functional groups: carboxyl and alcohol, obtaining an asymmetric carbon that provides it the optical activity. This acid has two optical isomers: lactic L (+) and lactic D (−) (Figure 1). However, only the L (+) isomer, which is considered a GRAS substance by the FDA, is considered a food additive e.g., as acidulant and preservative. However, it has other industrial importance in cosmetic, pharmaceutical, and chemical applications [12].

Figure 1.

Isomeric configurations of lactic acid [11].

Lactic acid has been widely used in recent decades, for example as the precursor of polylactic acid (a biodegradable biopolymer to manufacture packaging material for the food industry), 3D printing applications, and for medical uses, among others. Currently, the production of lactic acid is achieved through fermentation, however, the use of raw materials as sources of carbon and energy represents an environmental problem with a high cost. For this reason, environmentally friendly alternatives have been evaluated from agro-industrial by-products, as raw materials to obtain this acid [13]. For example, the use of whey cassava bark and pineapple bark, using immobilized Lactobacillus delbrueckii subsp. delbrueckii [14], Bioconversion of agroindustrial co-products into lactic acid by Latilactobacillus sakei [15]. Efficient conversion of agro-industrial waste into D (−) lactic acid with L. delbrueckii subsp. delbrueckii [16], the use of Lactobacillus, in bakery by-products, meat, whey, etc. [17]. In this chapter, the use of agro-industrial by-products is exposed through the use and application of LAB such as Lactobacillus in order to provide adding value to these interesting substrates.

2. Circular economy in agroindustry

In recent decades, the exploitation of natural resources and raw materials has increased, due to economic, productive, and environmental problems. These factors are decisive for the circular economy, which has forced it to change from a linear model to a circular model [18]. A linear economic model is based on the production, use, and disposal of a good or product. In the context of environmental sustainability, the circular economy proposes a strategy that reduces the negative impact on the environment where the final product is the source of value creation, increasing its useful life [19]. Circular economy is based on key strategies such as waste prevention design, where both, products and services are designed and created so that the production of waste is minimal or eliminated. Likewise, the idea is to give a second life to a product and thus reduce raw material to produce a new product [3]. Thus, the products must be more versatile, modular, and simple to adapt to different applications during their shell-life. In addition, it is important to use renewable energy to reduce the negative impact on the environment [3].

The benefits of the circular economy are linked to higher economic growth by using resources efficiently, creation of employment opportunities through the creation of industries that promote innovation and entrepreneurship, net savings in the cost of raw materials, reduction of environmental pollution in the form of carbon dioxide emission, reduction of water pollution and responsible use of the soil [20]. In 2015, the United Nations proposed the fulfillment of 17 Sustainable Development goals by 2030. To meet these objectives, there must be a joint action of governments, the private sector, civil society, etc. In the case of companies, it consists of promoting business models that are more committed to ecosystems, society, and living conditions of the world population [21].

3. Valorization of agro-industrial by-products using lactobacillus

At present, cellulosic materials are attractive as possible replacements for edible raw material made up of starch [22], materials such as agricultural residues are considered inexpensive and captivating materials to produce lactic acid since they are a great source of carbohydrates [23, 24]. The most used LAB to produce lactic acid by fermentation are Lactiplantibacillus plantarum, Amylolactobacillus. amylophilus, L, delbrueckii subsp. delbrueckii, L. delbrueckii subsp. bulgaricus and Lactobacillus acidophilus [25, 26, 27, 28]. For optimal lactic acid production it is necessary to make several considerations: strain of the microorganism with which to work, carbon source, temperature, pH, incubation time, and recycling of cells by immobilization [29]. For this reason, many attempts have been made to find low-priced substrates and interest has arisen in recycling agro-industrial by-products such as wheat bran, distillery residues, beer production residues, etc. These materials that are no longer used in different industries, could reduce the cost of lactic acid production, since raw materials such as starch and lignocellulosic materials require a physicochemical or enzymatic treatment for fermentations and that LAB can directly take advantage of these substrates [22].

For valorization of agro-industrial by-products using Lactobacillus, e.g., the production of lactic acid, several studies have been carried out with different types of substrates. For example, bakery waste with the microorganism Thermoanaerobacterium aotearoense [30], using whey with S. thermophillus and L. delbrueckii subsp. delbrueckii [31]. In addition, using cane bagasse as a substrate and Lactiplantibacillus pentosus [32], corn stubble has also been used to produce lactic acid by B. coagulans [33], coffee mucilage with L. delbrueckii subsp. bulgaricus [34], etc.

3.1 Whey

Today, the dairy industry is researching new technologies and looking for new products not only to meet consumer demand through innovative products but also to increase profitability. One of these alternatives is the addition of whey as a substitute for water [35]. However, some laws prohibit the sale of products derived from whey, to prevent food fraud.

Whey contributes with nutrients such as essential amino acids, in addition to the reduction of calories and improvements in technological properties [36]. The addition of this ingredient produces changes in the structure and in the case of chocolate drinks, it produces modifications in the functional and sensory properties [37].

The use of whey as an ingredient in food is regulated, for example, the FDA establishes a maximum of 5%. In white chocolate [38], a similar percentage of 5% of the total mass of chocolate has been established by the EU [39]. The use of Lactobacillus in whey as a substrate gives rise to new products, for example, enzymes with anti-hypertensive properties, which have been isolated after the fermentation of whey with Lactiplantibacillus plantarum QS670, Lactobacillus amylolyticus L6 has been used to prepare fermented tofu whey, it can be used as a starter culture to produce quagulant tofu and functional drinks [40].

Other studies indicate the suitability in the development of new foods based on lactic serum [41], whose fermentation converts its complex elements into simpler elements, facilitating assimilation in the intestinal tract [42]. Thus, the presence of probiotics increases the number of digestible amino acids, making fermented dairy products a good alternative as a source of nutrients, especially for diseases such as diarrhea [43].

An investigation ensure that salty whey fermented with the microorganism L. acidophilus 43S, fights harmful microorganisms such as Escherichia coli [6]. In addition, the fermentation of the whey favors the formation of peptides that can improve the functionality of food and beverages by reducing the taste of cheese that the whey has [17]. The way to obtain a probiotic drink based on milk serum is to drain, filter, and submit to a caloric process such as pasteurization, to later inoculate and ferment with the microorganisms of our choice. It is important to mention that the use of whey obtained by curd coagulation has an advantage over acid whey since, with sweet or deproteinized whey, clearer drinks are obtained without the formation of sediments.

3.1.1 Lactic acid and PLA made from whey

In the dairy industry, cheese is made by acidifying milk, which produces the precipitation of proteins known as casein, called curd. In this process, a by-product called whey is obtained, which contains other proteins such as lactalbumin, lactoferrin, and lactoglobulin, additionally, it has lactose, fat, and minerals such as calcium and iron that are present in milk [44].

Around 1/3 of the dairy production is destined for the production of cheese, of which between 80 and 90% of this volume corresponds to whey, which is considered a pollutant too, due to its high biological oxygen demand (BOD) and chemical oxygen demand (COD), currently, the treatment of waste from the food industry is mandatory and necessary to avoid damage to the environment [44].

Investigations have been carried out due to its multiple compounds and its ease of transformation into another useful product, to get the most out of the benefits it can provide. In the polymer industry, whey is used as a precursor for polylactic acid (PLA), a biodegradable material with uses in industry and medicine. Due to the scarcity and environmental regulations, alternatives for materials more compatible with the environment and independent of those of fossil fuels have been developed, this is why biopolymers such as PLA are adjusted to meet these needs in the industry, it is for the demand to elaborate more of these biopolymers has increased over time promoting a new industry worldwide, in the same way, other studies raise similar proposals with matrices other than PLA, focused mainly on starches and proteins [44].

As mentioned, lactic acid has two optical isomers D (−) and L (+), these can be obtained by fermentation using a LAB. However, by means of the chemical method, racemic DL-LA is always obtained [29]. The optical purity of lactic acid is of utmost importance within different industries that seek the combination of D (−) and L (+) polymers to obtain crystalline PLA with good mechanical properties [45]. There are several studies on the production of L (+) lactic acid; however, little research regarding the production of D (−) through fermentation processes were performed. Normally, the production of lactic acid occurs by batch, but this method has the disadvantage of reducing productivity and production due to inhibition by high concentrations of substrate, due to this, different alternatives have been reported in terms of improving productivity of lactic acid [46].

The effect of the different carbon sources for the development of the L. delbrueckii subsp. delbrueckii to the production of lactic acid using by-products like whey was evaluated [16]. Through this study, it was possible to conclude that the production of high optical purity D (−) lactic acid is possible by the microorganism, with the use of waste raw material such as molasses and corn liquor, without the need for pretreatment of these by-products. This represents a low cost to produce acid with high productivity using a fed-batch strategy [16].

The use of whey for the synthesis of polylactic acid, with the use of lactic acid bacteria, such as L. delbrueckii subsp. delbrueckii, through fermentation and its use as biodegradable material for food containers, is an alternative that must be studied yet. This proposal has great technological and environmental relevance because the development and characterization of the physical–chemical and mechanical properties of a biodegradable container from a by-product such as whey, will be able to solve contamination problems in the environment and the possibility of showing that natural polymers generate a greater contribution to the term of biodegradability [44].

3.2 Meat by-products

LAB is known to be present in the fermentation processes of meat products and by-products, to produce metabolites such as lactic and acetic acid. To carry out the fermentation of these meat products, it is possible to add an inoculum or simply take advantage of the bacterial flora present in the muscle fibers of the animal [17].

The microorganisms that are commonly found in animal meat and are involved in lactic fermentation are Pediococcus pentosaceus, Pediococcus acidilacti, Lactiplantibacillus plantarum. These microorganisms have beneficial effects on meat since there is the production of organic acids that favors the decrease in pH, and development of aroma and flavor, in addition to the partial denaturation of meat proteins, favoring the texture of these fermented products [47].

The decrease in pH helps with the elimination of harmful microorganisms that may be present in meat, especially in the viscera of the animal which are parts where pathogens prevail. The safe pH that the meat must reach is 4.0 to 4.2. However, in the case of fish this can take 48 hours to reach this point that, compared to poultry, their viscera reach the desired pH point between 24 to 36 hours [17].

3.3 Farinaceous by-products

Studies mention that to produce lactic acid it is necessary to use substrates rich in starch, such as starch from wheat, corn, rice, etc. [46]. However, these raw materials require pretreatments so that fermentation can be carried out, which is expensive for the industrial process. For this reason, alternatives have been found that can help in the production of this acid with the use of agro-industrial by-products such as molasses and corn liquor. These substrates must comply with the nutritional requirements of the LAB, and in this way reduce the cost of production [48]. For example, a study carried out to analyze and investigate the production of stereospecific lactic acid from agro-industrial by-products using strains belonging to Lactobacillus and Pediococcus in combination with enzymatic hydrolysis shown that raw materials, wheat bran (WB), distillery grains (DGS), used brewery grains (BSG) and lupine seeds (LF) (Lupinus angustifolius), can be efficiently synthesized [15]. In this investigation, favorable results were obtained for the propagation of LAB and the production of lactic acid. The lowest pH that was reached was with LF after 48 hours of fermentation with the strains of P. pentosaceus and Pediococcus acidilactici, while with the strain of Latilactobacillus sakei the substrate with the best result was WB. Thus, it is possible to conclude that with cellulosic agro-industrial waste using LAB, L-lactic acid can be efficiently synthesized [15].

Cassava (Manihot esculenta) is a tuber that has a large number of complex carbohydrates, which can lead to the production of lactic acid. This root is made up of 20% bagasse made up of peel and bark and 80% per tuber. Cassava bagasse is composed of 50 to 60% starch, approximately 34% cellulose, 15% hemicellulose, and 7% lignin [49]. In Colombia, studies have been carried out where the production of lactic acid was analyzed using a cassava starch solution and the use of Lactobacillus strains grown from yogurt [50]. A study with two different cassava varieties widely in Ecuador inoculated with L. delbrueckii subsp. lactis to produce lactic acid showed the sample with high starch presented highest lactic acid at pH of 5.5 and 150 rpm of stirring [51]. The results obtained in this study show that the production of lactic acid increases with pH with which it works during fermentation if the value ​​exceeds 5.5 there is a growth inhibition of lactic acid producing microorganisms from glucose [52]. The determining factors in this work were the substrate and the pH, it is also important to mention that the used microorganism directly consumes glucose to produce lactic acid; however, in the hydrolysis of starch, reducing sugars were obtained that include maltose, dextrins, and glucose. Regarding the pH and density, it was possible to determine a correlation between higher density values with the presence of lactic acid in the product obtained by fermentation, and consequently, the pH of this will be lower. Finally, it is possible to conclude that at higher pH with stirring, the microorganism works optimally in a fermentation process with hydrolyzed cassava bagasse, obtaining relevant yields. Thus, with the analyzes carried out on the lactic acid obtained, its quality was verified, and it may have value in the market for the sustainable development of Ecuador. However, the starch obtained from cassava bagasse for fermentation should be studied in greater depth, directly in the hydrolysis of the lignocellulosic material present [51].

Most of the biodegradable packaging developed for food use is based on the use of starches, among them those of cassava, potato, and achira, these by-products are subjected to fermentation by LAB to obtain lactic acid, precursor of PLA [51], also proteins such as zein are used, in both cases, the use of a source food to produce an inedible product poses a risk to food safety. There are few by-products used as raw material to produce biodegradable packaging, one of them is starch from cassava husk, there are no studies on the production of dairy packaging from PLA of whey from milk which must be inert and heat resistant [44].

Bakery waste is other product that can be fermented for obtain new foods, usually is used mainly to obtain breadcrumbs and for feed for livestock. It is possible to market it as a food with added value, for this, it is necessary to subject the by-products to crushing and drying to obtain a fine powder with low moisture content for these processes such as drying, mixing with other elements, and crushing is important [17]. Bakery by-products and other food waste have been subjected to fermentation to improve their physicochemical characteristics using LAB such as Ligilactobacillus salivarius [53]. The changes that can be seen due to anaerobic fermentation with LAB is the increase in soluble carbohydrates and nutritional improvement. However, it is achieved through a short fermentation of 10 days; thus, the microbial load is not so elevated. To carry out a fermentation process like this, it was considered that 0.2% inoculum is the optimal amount for improving the nutritional properties of bakery coproducts using LAB [47].

3.4 Fruit by-products

To produce lactic acid, the use of fruit waste as liquid pineapple could be beneficial since it is an element rich in glucose and nutrients [14]. The pineapple canning industry is one of the food industries that generates a large amount of both solid and liquid waste and that must follow strict environmental regulations. For this reason, there is a special interest in this productive sector. In addition, the use of effluents as carbon sources for lactic acid fermentation helps to reduce or eliminate pollution and reduce costs [14]. However, there is the presence of metals such as copper, zinc, magnesium, calcium, iron, etc., which can cause problems in the fermentation to produce lactic acid. These can inhibit the growth of LAB, influence the pH of the substrate, and also be involved with the inactivation of enzymes that participate in the synthesis of products [26]. Currently, the most widely used microorganism immobilization matrix has been sodium alginate, this matrix has been used for Saccharomyces cerevisiae, Bacillus amyloliquefaciens, and Kluyveromyces. The advantage of the use of this matrix is its stability; The substrates and products easily diffuse in and out. However, there is much work on the production of lactic acid, the use of pineapple residues and the immobilization of lactic microorganisms have not been explored [54]. In this study, it was found that sodium alginate in a concentration of 2% generates the maximum production of lactic acid in comparison with the other concentrations tested. Regarding pH, the best yield in the production of lactic acid was 6.5 and finally, the optimal temperature during fermentation was 37°C [14]. Thus, it is possible to conclude that the production of lactic acid using effluents from the pineapple canned industry is viable, if the optimal sodium alginate conditions, the working pH, and the temperature are considered [14].

3.5 Marine by-products

The main elements of interest from shellfish by-products are pigments such as carotenoids and melanin. Carotenoids are responsible for the coloring of the meat and skin of some fish such as salmonids, they have warm colors such as yellow, orange, and red, Furthermore, they are found in the exoskeletons of crustaceans such as crabs, lobsters, and shrimp. Melanin is a dark pigment that ranges from brown to black, this product of an oxidation reaction of phenolic compounds is present in the peritoneal lining, skin, and eyes of some species [17].

For the extraction of carotenoids from the by-products of crustaceans, several studies have been carried out, using enzymatic or fermentative methods, that are more preferable for compounds such as carotenoids, thus, obtaining higher yields and higher quality carotenoids for their possible applications [17]. Regarding very unstable carotenoids, the silage method by fermentation with lactic acid has been tried, in order to stabilize astaxanthin [55]. The extraction of carotenoids by means of silage of shrimp by-products (L. delbrueckii subsp. indicus) by Lactiplantibacillus plantarum was studied, in addition, extraction with a mixture of hexane and isopropanol as solvents and refined sunflower oil and the effect of each of the procedures to analyze the stability of the carotenoids, obtaining that the fermentation turned out to be better compared to acid silage, in solvent as in oil [17].

4. Conclusion

The use of agro-industrial by-products with LAB, specifically Lactobacillus spp., and consequently the production of lactic acid, fits within the ideology and concept of circular economy, focusing on the pillars of economic, ecological, and social aspects. The use of material that is usually discarded represents a relief for the planet in terms of the reduction of polluting matter that thanks to innovation can be used, the reduction of costs for companies by substituting more expensive raw material for another more economically accessible, and the social contribution through the generation of employment and economic activation [51]. This favorable impact, sought by the United Nations with the fulfillment of the Sustainable Development goals proposed for the year 2030, can also be reflected in the valorization of agro-industrial by-products not only to produce lactic acid but also in other applications. Examples of these applications are nutraceutical formulations and pharmaceuticals through the use of fungal co-products [56], food packaging through the use of whey [57], valorization of lard through biotechnological tools employing fermentation in state solid of Yarrowia lipolytica and Lacticaseibacillus paracasei [58], biorefining of whey from cheese in order to generate high-value products and eliminate environmental contamination by whey [59], revaluation of by-products of the wine industry through the application of LAB strains [60], use of fruit and vegetable by-products for the development of pharmaceutical products [61], etc.