MATERIALS & METHODS
Author highly acknowledges Yobe State University, Damaturu, Yobe State, Nigeria for providing moral support for this research work.
Reader & Head
Department of Biological Sciences
Yobe State University
Damaturu, Yobe State, Nigeria
Cheese whey is an important by product from the cheese manufacturing industry. Typically, 100g of milk yields 10g of cheese and 90g of liquid whey. Cheese whey contains about 4.5-5% lactose, 0.6-0.8% soluble protein, 0.4-0.5%(w/v) lipids, and varying concentrations of mineral salts. Cheese whey disposal has long been a problem for the dairy industry. Most medium and small cheese producers still dispose of their whey or whey permeates directly on farmland, which can pose an environmental risk. In response, regulations for land spreading of cheese whey are tightening and looking to ban land spreading of cheese whey. Ultra filtration has been used to separate whey protein from lactose sugar and other components in the whey. Whey protein has found a good market as a food additive or protein supplement.
Lactic acid is a natural organic acid and has many applications in the pharmaceutical, food, and chemical industries. It is used as acidulate and preservative, and recently, its potential as substrate for the production of biodegradable plastic has been actively pursued. Approximately half of the world’s supply of lactate is produced by fermentation process. Lactic acid has been produced by fermentation of sugar-containing substrates including cheese whey using lactococcus lactic.
Extensive research has been attributed to the sequencing of Lactococcus lactis genome due to its major role in the production of dairy products and preservation. Manufactures use the discovered properties of L. lactis to increase food preservation, distinguish flavor and aroma. L. lactis contains a bacteriocin, called nisin. It is a natural antimicrobial agent that fights against a wide range of Gram-positive bacteria, such as food-borne pathogens. Uses of nisin, such as controlling spoilage in lactic acid bacteria, have been present in alcohol production, wine, beer, and high acidic foods such as salad dressings.
L. lactis holds great value to the dairy product industry. The bacterium is found in milk and its main function is to produce lactic acid, which improves preservation needed for over nine million pounds of cheese produced each year. The bacterium can be a single strain starter culture or a mixed strain culture with other lactic acid bacteria. It is a key starter culture in the production of varies types of cheese such as cheddar, Colby, cottage cheese, cream cheese, as well as other dairy products like cultured butter, buttermilk, and sour cream. The cheese producing industry contributes greatly to the country’s economy. In Wisconsin alone 2.5 billion lbs of cheese are produced annually and 90% of their milk is used to produce cheese. Wisconsin's cheese producing revenue has accumulated up to $18 billion a year. The cheese industry is of such great importance in Wisconsin, that it has nominated Lactococcus lactis as the state microbe.
Yeast extract was commonly recommended as the best source for growth stimulation and lactic acid production by lactic acid bacteria. Lactic acid can be produced on whey permeate by batch cultivation, continuous process, or by immobilized cell process.
The batch process is the most studied and most commonly used method for lactic acid production on whey permeates. To prevent biosynthesis inhibition by the accumulated product, pH must be maintained at the optimum level of 6.0. The free lactic acid concentration in that case will be approximately 0.3g/L, which is below the inhibitory level. pH can be maintained by adding sodium hydroxide, ammonium hydroxide, or calcium ions.
Lactic acid is an organic acid (a-hydroxy-propionic acid) which can be used for a wide variety of industrial applications. In food industry it is used as acidulate, Preservative and antimicrobial agent For pharmaceutical applications, lactic acid can be used as electrolytes and mineral sources. For technical applications lactic acid can be used as neutralizers, solvents, cleaning agents, slow acid release agents and metal complexing agents. It has also been used in cosmetic industry as pH buffer, antimicrobial, skin rejuvenating and skin lightening.
There are two isomers of lactic acid, these are D(-) and L(+) forms. They only Differ in their optical properties, but are identical in their physical and chemical Characteristics. L(+)-lactic acid is biodegradable and can be metabolized by the human body and this property leads the application of lactic acid in biomaterial and biomedical field.
Lactic acid is produced chemical synthesis and by microbial fermentation. By Chemical synthesis method, racemic (DL) mixture of lactic acid is produced. By Microbial fermentation method L(+) and D(-) lactic acids can be produced according to the type of microorganism which may be homofermentative or heterofermentative. This is an important advantage of the microbial fermentation method compared to the chemical synthesis method. At the end of the fermentation process, lactic acid exists in the complex medium of fermentation broth which contains whey proteins, biomass, salts and other impurities. Lactic acid should be recovered from that complex media. As high cost of lactic acid purification process limits the utilization of this chemical, in large scale applications a system with less raw material and fewer unit operations are needed.
Whey is a major by-product of the dairy industry which serves as an inexpesive medium for lactic acid production. It contains approximately (w/v) 5 % lactose, 1 % protein, 0.4 % fat, and some minerals. It has a high biochemical oxygen demand (BOD) content (40,000-60,000 ppm) which represents serious disposal problems .
Physical and Chemical Properties of Lactic Acid
Lactic acid was first isolated from sour milk by Scheele in 1780. Lactic acid exists in two optically active isomeric forms as shown in Figure. 1
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D(-) and L(+) lactic acids differ in their optical properties, but are identical in their physical and chemical characteristics.
Lactic acid may react as an organic acid as well as an organic alcohol and can Participate in numerous types of chemical reactions. Some characteristics of lactic acid organisms are shown in Table.1
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Although the L (+) form appears to be dextrorotatory, it may actually be levorotatory as are its salts and esters. The apparent reversal in optical rotation May be due to the formation of an ethylene oxide bridge between carbon atoms 1 and 2 bytautomeric shift of the hydroxyl group on carbon atom 2 to the carbonyl group of the carboxyl radical. Salts and esters of L(+)- lactic acid cannot form this epoxies ring and are levorotatory.
Lactic acid is soluble in all proportions of water and exhibits a low volatility. In solutions with roughly 20 % or more lactic acid, self-etherification occurs because of the hydroxyl and carboxyl functional groups. Lactic acid may form a cyclic dimmer (lactide) or form linear polymers with the general formula
H [OCH(CH3)CO]n OH.
Application of Lactic Acid
Lactic acid is sold in food, pharmaceutical and technical grades. Since the lactic acid has gained increasing importance and has been used in a great variety of applications, its salt, ester and many derivatives have been developed. The uses of lactic acid can be broken down by grade and by lactic acid derivatives. Some of the important applications of lactic acid are detailed below.
Lactic acid is used in pharmaceutical industry as a very important ingredient. Pharmaceutical and food industries show presence for the L(+)lactic acid because the D(-) isomer is not metabolized by the human body. Lactic acid and its salts have been mentioned for various medical uses. They provide the energy and volume for blood besides regulation of pH Calcium, sodium, ferrous and other salt of lactic acid are used in pharmaceutical industry in various formulations find use for their anti tumour activity. Lactic acid finds medical applications as an intermediate for pharmaceutical manufacture, for adjusting the pH of preparations and in tropical wart medications .
Biodegradable plastic made of poly (lactic acid) is used for sutures that do not need to be removed surgically and has been evaluated for use as a biodegradable implant for the repair of fractures and other injuries. These applications can be divided into:
- Bone implants
- Ca-lactate in calcium tablets
- Co-polymers in controlled drug release
- Sodium lactate in dialysis solutions
- Skin and hair care (cosmetics industry)
- Lactic acid (skin renewal process)
- Sodium and ammonium-lactate (skin moisturizer)
- Hair conditioner
The calcium salts of lactic acid are produced in a granular and powdered form. Calcium lactate tri hydrate is used in pharmaceuticals primarily as a dietary calcium source and also as a blood coagulant for use in the treatment of haemorrhages and to inhibit bleeding during dental operations. Sodium lactate is used in the production of some antibiotics and to buffer pharmaceutical preparations.
Natural L (+) lactic acid is used in many applications in cosmetics. Lactic acid is an alpha hydroxyl acid and is found in the skin. It is used as a skin-rejuvenating agent, pH regulator. It is a common ingredient in moisturizers, skin whiteners and anti acne preparation. Since L (+) Lactic acid is naturally present in the skin, lactic acid and sodium lactate are extensively used as moisturizing agents in many skin care products. Lactic acid is also used as a pH-regulator. It is one of the most effective AHAs and has the lowest irritation potential. Lactates are regarded as skin whitening agents that have been shown to produce a synergistic effect when combined with other skin whitening agents .
Lactic acid occurs naturally in many food products. It has been in use as an acidulate, preservative and pH regulator for quite some time. Some of the important applications of lactic acid in the food industry are detailed below. There are many properties of lactic acid, which make it a very versatile ingredient in the food industry. It has a pronounced preservative action, and it regulates the micro flora. It has been found to very effective against certain type of microorganisms. Sometimes a combination of lactic acid and acetic acid is used as it has a greater bactericidal activity. Because it occurs naturally in many food stuffs, it does not introduce a foreign element into the food. The salts are very soluble, and this gives the possibility of partial replacing the acid in buffering the acid in buffering systems .
Lactic acid is non-toxic and is deemed “Generally Recognized As Safe” (GRAS) as a general-purpose food additive in the USA. The same status is accorded in many other countries too. The calcium salt of lactic acid, calcium lactate, has greater solubility than the corresponding salt of citric acid. In such products, where turbidity caused by calcium salts is a problem, the use of lactic acid gives products, which are clear. L(+) Lactic acid is the natural lactic acid found in biological systems and hence its use as an acidulate does not introduce a foreign element into the body. Lactic acid are widely used in food industry such as confectionery as acidulate, beverages industries as natural flavoring, a preservatives for fermented vegetable and meat, and also an vital element for producing dairy’s product.
Direct acidification with lactic acid in dairy products such as cottage cheese is preferred to fermentation as the risks of failure and contamination can be avoided. The processing time also can be saved. Lactic acid is also used as an acidulate in dairy products like cheese and yogurt powder. The production of processed cheese can be greatly simplified if a sufficient amount of lactic acid is added to the freshly drained cheese curd to lower the pH to 4.8-5.2, then the curd can be processed without further curing, to adjust acidity and improved flavor, texture and stability.
The technical uses for lactic acid comprise a relatively small portion of the world’s production. These applications can be divided into:
- Lactate esters in solvents photo resist formulations
- Solder flux remover
- Replacing ozone-depleting solvents
- Degreasing/ cleaning of metal surfaces
- Coating and ink
- Cataphoretic electro-deposition coating (acid)
- Solvent for coating and ink (ester)
- Polylactic acid (PLA)
In the United State, Europe and Japan, several companies are actively pursuing development and commercialization of polylactic acid products. PLA polymers can be synthesized from various monomers. Low molecular weight polymers are obtained by step-growth polymerization of lactic acid. Whereas high molecular weight polymers are synthesized by ring-opening polymerization of lactide as shown in Figure 2.2. Lactide is composed of two lactic acid units linked to form a diester cyclic monomer. Step growth polymerization of optically pure L-lactic acid (or pure D-lactic acid) and ring opining polymerization of optically pure L-lactide (or pure D-lactide) should lead to the same chain growth.
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Figure. 2 Synthesis of PLA using ring-opening polymerization
Actually dramatic differences in main chain structures are observed as soon as one deals with stereo copolymers of L-and D-lactic acid repeating units. The step growth polymerization of mixtures of L- and D-lactic acid leads to poly (D,L-lactic acid) with a random distribution of the L- and D-lactyl units, whereas ring opening polymerization of the lactide dimmers lead to non-random distribution because chains grow through a pair addition mechanism. The difference in the crystalline of poly (D, L-lactic acid) and poly (L-lactic acid) has important practical ramifications. Since poly (D, L-lactic acid) is an amorphous polymer; it is usually considered for applications such as drug delivery where it is important to have homogenous dispersion of the active species within a monophonic matrix. On the other hand, the semi crystalline poly (L-lactic acid) is preferred in applications where high mechanical strength and toughness is required (i.e. sutures and orthopedic devices).
PLA polymers offer a broad balance of functional performance that makes them suitable for a wide variety of market applications. They are expected to compete with hydrocarbon-based thermoplastics on a cost or performance basis. It also exhibits a tensile strength and modulus comparable to some thermoplastics. Like PET (polyethylene terephthalate), these polymers resist grease and oil and offer good flavor and odor barrier. PLA polymers also provide for heat stability at lower temperature than polyolefin sealant resin. The polymer can be processed by most melt fabrication techniques including thermoforming, sheet and film extrusion, blown film processing, fiber spinning and injection molding.
This material biodegrades completely to carbon dioxide and water when composted in municipal or industrial facilities, unlike traditional degradable plastics that need ultraviolet radiation to degrade. PLA needs only water and thus will degrade in the landfills. Biodegradation of PLA proceeds by a two-step process. Initially hydrolysis produces progressive chain length reduction by what is essentially an ester interchange process. This reaction is catalyzed by heat and pH. There are no bacteria involved in this phase of the process. When the chain length is reduced, producing very low molecular weight fragments, naturally occurring bacteria digest residues and liberate carbon dioxide and water. Considering the above facts research studies were carried out on production of lactic acid utilizing low-cost fermentation medium, such as Dairy wastes.
The compound was first identified by an eighteenth century Swedish chemist named Carl Wilhelm Scheele. With the advancement in biological sciences man has realized the importance of Bioconversion of industrial wastes into value added products through the knowledge of sweeping role of microorganisms. Wastes are frequently used as Substrates in fermentation as an inexpensive source. Dairy wastes are used for the production of organic acids, which are widely used as food preservatives/additives. The available literature on various aspects of organic acids production through fermentation technology while using dairy wastes as substrates is reviewed under the following headings:
Lactic acid (2-hydroxypropionic acid) was initially isolated from sour milk in 1789 by Scheele and received its name from milk sugar (lactose) fermented by Streptococcus lactis. It was the first organic acid to be produced at an industrial scale by fermentation in 1880. Through controlled fermentation, lactic acid was manufactured from the hexose sugars, molasses, corn, or milk. Since 1930, lactic acid has been commercially produced from whey, a by-product from milk. Lactic acid (2-hydroxypropionic acid) was initially isolated from sour milk in 1789 by Scheele and received its name from milk sugar (lactose) fermented by Streptococcus lactis. It was the first organic acid to be produced at an industrial scale by fermentation in 1880. Through controlled fermentation, lactic acid was manufactured from the hexose sugars, molasses, corn, or milk. Since 1930, lactic acid has been commercially produced from whey, a by-product from milk.
Lactic acid has many applications in food, pharmaceutical, leather, and textile industries . Since lactic acid has high reactivity due to both hydroxyl (-OH) and carboxyl (-COOH) group. It plays a major role as a chemical feedstock capable of being converted to various chemicals such as acrylic acid, propylene glycol, acetaldehyde, and 2, 3-pentanedione .
The continuous increase in demand for lactic acid has been due to its increasing applications in preparation of biodegradable polymers and green solvents.
Lactic acid has long been used in the pharmaceutical, chemical, and especially in the food industry as an acidulate, taste-enhancer and a preservative. It is used as a raw material for the formation of polylactic acid (PLA) for the manufacture of new biodegradable plastics. Indeed this opens up new opportunities in the plastic industry with the reduction, and possibly the elimination, of non-biodegradable plastic wastes,.
The homo fermentative microorganisms produce no carbon dioxide from sugars, and thus more lactic acid is synthesized. It is most feasible to obtain strains of lactic acid bacteria producing only L(+) or D(-) lactic acid. L (+)-Lactic acid is commercially produced by fermentation processes using lactic acid bacteria or fungi such as Rhizopus in submerged culture. but the production yield based on total carbohydrate consumed by Rhizopus oryzae is low in comparison to lactic acid bacteria. Nevertheless, Rhizopus arrhizus and R. oryzae are widely accepted producers of lactic acid. The L(+)-lactic acid obtained from R. oryzae has a high optical purity which is required in the starting material for PLA production, in order to obtain high quality crystal biopolymers.
In an experiment five strains of Bacillus via; Bacillus cereus JCM 2152, Bacillus coagulants JCM 2257, Bacillus subtilus JCM 1465, Bacillus thuringiensis subsp. Kurstaki ATCC 33679, and strain SHO-1 and eight strains of Rhizopus viz; R. oryzae IFO 31005, IFO4707, IFO4798, IFO5380, IFO5384, and NRRL395; and R. chinensis F-595 were used in simple media to produce lactic acid. The R. oryzae NRRL395 produced 100% L (+)-form lactic acid with the highest yield, which was considered ideal for the manufacture of PLA. The presence of lactic acid with high optical purity gives polylactic acids of high melting point and high crystalline.