FOOD AND INDUSTRIAL MICROBIOLOGY

Module 9. Microbial process strategies

Lesson 28
ORGANIC ACIDS – CITRIC, LACTIC AND VINEGAR

28.1 Introduction

A large number of organic acids with actual or potential uses are produced by microorganisms. Citric, itaconic, lactic, mallic, tartaric, gluconic, mevalonic, salicyclic, gibberelic, diamino-pimelic, and propionic acids are some of the acids produced using microorganisms.

28.2 Production of Citric Acid

Citric acid is a tribasic acid. It crystallizes with large rhombic crystals containing one molecule of water of crystallization, which is lost when it is heated to 130°C. At temperatures as high as 175°C it is converted to itaconic acid, aconitic acid, and other compounds.

28.2.1 Uses of citric acid

Citric acid is used in the food industry, in medicine, pharmacy and in various other industries.

Uses in the food industry

(i) Citric acid is the major food acidulant used in the manufacture of jellies, jams, sweets, and soft drinks.

(ii) It is used for artificial flavoring in various foods including soft drinks.

(iii) Sodium citrate is employed in processed cheese manufacture.

Uses in medicine and pharmacy

(iv) Sodium citrate is used in blood transfusion and bacteriology for the prevention of blood clotting.

(v) The acid is used in effervescent powers which depend for their effervescence on the CO₂ produced from the reaction between citric acid and sodium bicarbonate.

(vi) Since it is almost universally present in living things, it is rapidly and completely metabolized in the human body and can therefore serve as a source of energy.

Uses in the cosmetic industry

(vii) It is used in astringent lotions such as aftershave lotions because of its low pH.

(viii) Citric acid is used in hair rinses and hair and wig setting fluids.

Miscellaneous uses in industry

(ix) In neutral or low pH conditions the acid has a strong tendency to form complexes hence it is widely used in electroplating, leather tanning, and in the removal of iron clogging the pores of the sand face in old oil wells.

(x) Citric acid has recently formed the basis of manufacture of detergents in place of phosphates, because the presence of the latter in effluents gives rise to eutrophication (an increase in nutrients which encourages aquatic flora development).

28.2.2 Biochemical basis of the production of citric acid

Citric acid is an intermediate in the citric acid cycle. The acid can therefore be caused to accumulate by one of the following methods:

(a) By mutation – giving rise to mutant organisms which may only use part of a metabolic pathway, or regulatory mutants; that is using a mutant lacking an enzyme of the cycle.

(b) By inhibiting the free-flow of the cycle through altering the environmental conditions, e.g. temperature, pH, medium composition (especially the elimination of ions and cofactors considered essential for particular enzymes). The following are some of such environmental conditions which are applied to increase citric acid production:

(i) The concentrations of iron, manganese, magnesium, zinc, and phosphate must be limited.

(i) The dehydrogenases, especially isocitrate dehydrogenase, are inhibited by anaerobiosis, hence limited aeration is done on the fermentation so as to increase the yield of citric acid.

(iii) Low pH and especially the presence of citric acid itself inhibits the TCA and hence encourages the production of more citric acid.

Citric acid can be caused to accumulate by using a mutant lacking an enzyme of the cycle or by inhibiting the flow of the cycle.

28.2.3 Fermentation for citric acid production

For a long time the production of citric acid has been based on the use of molasses and various strains of Aspergillus niger and occasionally Asp. wenti. Although several reports of citric acid production by Penicillium are available, in practice, organisms in this group are not used because of their low productivity. In recent times yeasts, especially Candida spp. (including Candida quillermondi) have been used to produce the acid from sugar.

(a) Surface fermentation: Surface fermentation using Aspergillus niger may be done on rice bran as is the case in Japan, or in liquid solution in flat aluminium or stainless steel pans. Special strains of A. niger which can produce citric acid despite the high content of trace metals in rice bran are used. The citric acid is extracted from the bran by leaching and is then precipitated from the resulting solution as calcium citrate.

Fig. 28.1 Citric acid cycle (Prescott, 2000)

(b) Submerged fermentation: As in all other processes where citric acid is made the fermentation the fermentor is made of acid-resistant materials such as stainless steel. The carbohydrate sources are molasses decationized by ion exchange, sucrose or glucose. The pH is never allowed higher than 3.5. Copper is used at up to 500 ppm as an antagonist of the enzyme aconitase which requires iron. 1-5% of methanol, isopranol or ethanol when added to fermentations containing unpurified materials increases the yield; the yields are reduced in media with purified materials. As high aeration is deleterious to citric acid production, mechanical agitation is not necessary and air may be bubbled through. Antifoam is added. The fungus occurs as a uniform dispersal of pellets in the medium. The fermentation lasts for five to fourteen days.

28.2.4 Extraction

The broth is filtered until clear. Calcium citrate is precipitated by the addition of magnesium-free Ca(OH)₂. Since magnesium is more soluble than calcium, some acid may be lost in the solution as magnesium citrate if magnesium is added. Calcium citrate is filtered and the filter cake is treated with sulfuric acid to precipitate the calcium. The dilute solution containing citric acid is purified by treatment with activated carbon and passing through iron exchange beds. The purified dilute acid is evaporated to yield crystals of citric acid.

28.3 Lactic Acid

Lactic acid is produced by many organisms: animals including man produce the acid in muscle during work.

28.3.1 Properties and chemical reactions of lactic acid

(i) Lactic acid is a three carbon organic acid: one terminal carbon atom is part of an acid or carboxyl group; the other terminal carbon atom is part of a methyl or hydrocarbon group; and a central carbon atom having an alcohol carbon group.

(ii) Lactic acid is soluble in water and water miscible organic solvents but insoluble in other organic solvents.

Technical grade lactic acid is used as an acidulant in vegetable and leather tanning industries. Various textile finishing operations and acid dyeing of food require low cost technical grade lactic acid to compete with cheaper inorganic acid. Lactic acid is being used in many small scale applications like pH adjustment, hardening baths for cellophanes used in food packaging, terminating agent for phenol formaldehyde resins, alkyl resin modifier, solder flux, lithographic and textile printing developers, adhesive formulations, electroplating and electropolishing baths, detergent builders. Lactic acid has many pharmaceutical and cosmetic applications and formulations in topical ointments, lotions, anti acne solutions, humectants, parenteral solutions and dialysis applications, and anti carries agents. Calcium lactate can be used for calcium deficiency therapy, and as an anti caries agent. Its biodegradable polymer has medical applications as sutures, orthopedic implants, controlled drug release, etc. Polymers of lactic acids are biodegradable thermoplastics. These polymers are transparent and their degradation can be controlled by adjusting the composition, and the molecular weight.Their properties approach those of petroleum derived plastics. Lactic acid esters like ethyl/butyl lactate can be used as green solvents. They are high boiling, non-toxic and degradable components. Poly L-lactic acid with low degree of polymerization can help in controlled release or degradable mulch films for large-scale agricultural applications. Lactic acid was among the earliest materials to be produced commercially by fermentation and the first organic acid to be produced by fermentation.

28.3.2 Fermentation for lactic acid

Although many organisms can produce lactic acid, the amount so produced is small: the organisms which produce adequate amounts and are therefore used in industry are the homofermentative lactic acid bacteria, Lactobacillus spp., especially L. delbruckii. In recent times Rhizopus oryzae has been used. Both organisms produce the L- form of the acid, but Rhizopus fermentation has the advantage of being much shorter in duration; further, the isolation of the acid is much easier when the fungus is used.

Lactic acid is very corrosive and the fermentor, which is usually between 25,000 and 110,000 liters in capacity, is made of wood. Alternatively special stainless steel (type 316) may be used. They are sterilized by steaming before the introduction of the broth as contamination with thermophilic clostridia yielding butanol and butyric acid is common. Such contamination drastically reduces the value of the product. During the step-wise preparation of the inoculum, which forms about 5% of the total beer, calcium carbonate is added to the medium to maintain the pH at around 5.5-6.5. The carbon source used in the broth has varied widely and has included whey, sugars in potato and corn hydrolysates, sulfite liquour, and molasses. However, because of the problems of recovery for high quality lactic acid, purified sugar and a minimum of other nutrients are used.

Lactobacillus requires the addition of vitamins and growth factors for growth. These requirements along with that of nitrogen are often met with ground vegetable materials such as ground malt sprouts or malt rootlets. To aid recovery the initial sugar content of the broth is not more than 12% to enable its exhaustion at the end of 72 hours. Fermentation with Lactobacillus delbruckii is usually for 5 to 10 days whereas with Rhizopus oryzae, it is about two days. Although lactic fermentation is anaerobic, the organisms involved are facultative and while air is excluded as much as possible, complete anaerobiosis is not necessary. The temperature of the fermentation is high in comparison with other fermentation, and is around 45°C. Contamination is therefore not a problem, except by thermophilic Clostridia.

28.3.3 Extraction

The main problem in lactic acid production is not fermentation but the recovery of the acid. Lactic acid is crystallized with great difficulty and in low yield. The purest forms are usually colorless syrups which readily absorb water. At the end of the fermentation when the sugar content is about 0.1%, the beer is pumped into settling tanks. Calcium hydroxide at pH 10 is mixed in and the mixture is allowed to settle. The clear calcium lactate is decanted off and combined with the filtrate from the slurry. It is then treated with sodium sulfide, decolorized by adsorption with activated charcoal, acidified to pH 6.2 with lactic acid and filtered. The calcium lactate liquor may then be spray-dried.

For technical grade lactic acid the calcium is precipitated as CaSO4.2H2O which is filtered off. It is 44-45% total acidity. Food grade acid has a total acidity of about 50%. It is made from the fermentation of higher grade sugar and bleached with activated carbon. Metals especially iron and copper are removed by treatment with ferrocyanide. It is then filtered. Plastic grade is obtained by esterification with methanol after concentration. High-grade lactic acid is made by various methods: steam distillation under high vacuum, solvent extraction etc.

28.4 Vineger

Vinegar is a product resulting from the conversion of alcohol to acetic acid by acetic acid bacteria, Acetobacter spp. The name is derived from French (Vin = wine; Aigre-sour or sharp). Although acetic acid is the major component of vinegar, the material cannot be produced simply by dissolving acetic acid in water. When alcoholic fermentation occurs and later during acidifications many other compounds are produced, depending mostly on the nature of the material fermented and some of these find their way into vinegar. Furthermore, reactions also occur between these fermentation products. Ethyl acetate, for example, is formed from the reaction between acetic acid and ethanol. It is these other compounds which give the various vinegars their bouquets or organoleptic properties. The other compounds include non-volatile organic acids such as malic, citric, succinic and lactic acids; unfermented and unfermentable sugars; oxidized alcohol and acetaldelyde, acetoin, phosphate, chloride, and other ions.

28.4.1 Uses

(i) Ancient uses : The ancient uses of vinegar which can be seen from various records include a wide variety of uses including use as a food condiment, treatment of wounds, and a wide variety of illnesses such as plague, ringworms, burns, lameness, variocose veins. It was also used as a general cleansing agent. Finally, it was used as a cosmetic aid.

(ii) Modern uses: Vinegar is used today mainly in the food industry as; (a) a food condiment, sprinkled on certain foods such as fish at the table; (b) for pickling and preserving meats and vegetables; vinegar is particularly useful in this respect as it can reduce the pH of food below that which even sporeformers may not survive; (c) It is an important component of sauces especially renowned French sauces many of which contain vinegar; (d) Nearly 70% of the vinegar produced today is supplied to various arms of the food industry where it finds use in the manufacture of sauces, salad dressings, mayonnaise, tomato productions, cheese dressings, mustard, and soft drinks.

28.4.2 Substrate

The substrate for the alcoholic fermentation for vinegar productions varies from one locality to the other. Thus, while wine vinegar made from grapes is common in continental Europe and other vine growing countries, malt vinegar is common in the United Kingdom; the United States on account of its great variety of climatic regions uses both malt and wine vinegars. Rice vinegar is common in the far Eastern countries of Japan and China and pineapple vinegar is used in Malaysia. In some tropical countries vinegar has been manufactured from palm wine derived from oil or raffia palm.

28.4.3 Organisms involved

Although Acetobacter spp are responsible for vinegar production, pure cultures are hardly used, except in submerged fermentation because of the difficulty of isolating and maintaining the organisms. The only member of the genus which is not useful, if not positively harmful in vinegar production is Acetobacter xylinum which tends to produce slime. Recently a new species, Acetobacter europaeus, was described. Its distinguishing features are its strong tolerance of acetic acid and its absolute requirement of acetic acid for growth.

Strains of acetic acid bacteria to be used in industrial production should a) tolerate high concentrations of acetic acid; b) require small amounts of nutrient; c) not overoxidize the acetic acid formed; and d) be high yielding in terms of the acetic acid produced.

The biochemical processes are simple and are shown below:

28.4.4 Manufacture of vinegar

The three methods used for the production of vinegar are the Orleans Method (also known as the slow method), the Trickling (or quick) Method and Submerged Fermentation. The last two are the most widely used in modern times.

The common feature in all submerged vinegar production is that the aeration must be very vigorous as shortage of oxygen because of the highly acid conditions of submerged production, would result in the death of the bacteria within 30 seconds. Furthermore, because a lot of heat is released (over 30,000 calories are released per gallon of ethanol) an efficient cooling system must be provided. All submerged vinegar is turbid because of the high bacterial content and have to be filtered.

28.4.5 Frings acetator

First publicized in 1949, most of the world’s vinegar is now produced with this fermentor. It consists of a stainless steel tank fitted with internal cooling coils and a highspeed agitator fitted through the bottom. Air is sucked in through an air-meter located at the top. It is then finely dispersed by the agitator and distributed throughout the liquid. Temperature is maintained at 30°C, although some strains can grow at a higher temperature. Foaming is interrupted with an automatic foam breaker. Essentially it is shaped like the typical aerated stirred tank fermentor. It is operated batchwise and the cycle time for producing 12% vinegar is about 35 hours. The Frings alkograph automatically monitors the alcohol content and signals the end of the batch when the alcohol content falls to 0.2% (v/v). At this stage about one third of the product is pumped out and fresh feed pumped to the original level. The aeration must continue throughout the period of the unloading and loading. A fermentation cycle takes 24 to 48 hours Since its first description, improvements and modifications have been made on the Frings acetator.

28.4.6 Processing of vinegar

(a) Clarification and bottling: Irrespective of the method of manufacture, vinegar for retailing is clarified by careful filtration using a filter aid such as diatomaceous earth. Vinegar from trickling generators are however less turbid than those from submerged fermentations because a high proportion of the bacterial population responsible for the acetification is held back on the shavings. After clarification it is pasteurized at 60-65°C for 30 minutes.

(b) Concentration of vinegar: Vinegar can be concentrated by freezing; thereafter the resulting slurry is centrifuged to separate the ice and produce the concentrate. With this method 200° grain (i.e., 20% w/v) acetic acid can be produced.