CHAPTER V: Industrial fermentation products

Microorganisms are produced for their cells themselves or for the contents extracted from these cells and can therefore serve as food, they can also produce flavoring agents or flavor enhancers and contribute to the quality of food. Food supplements obtained by fermentations can serve as preservatives or agents to increase the nutritional value of the food, or also to convert basic foods such as milk or meat into more elaborate products.

V.1. Proteins of Single Cell Origin (P.O.U).

   V.1.1. Definition.

We call “Proteins of Unicellular Origin” (P.O.U), or in English “Single Cell Proteins (SCP)”, any microbial biomass rich in proteins intended for human or animal food.

P.O.U are not pure proteins, but also contain carbohydrates, lipids, nucleic acids, mineral salts and vitamins.

The motivation for the use of P.O.U in human food is mainly aimed at overcoming undernutrition in countries with food difficulties, and therefore makes it possible to satisfy their protein needs. In countries where there are no serious problems of undernutrition, the use of P.O.U in human food is very limited, and it is rather intended for animal feed.

Probiotics are bacteria or yeasts that play an important role in digestion or immune defense. For this, probiotics are consumed in the form of food supplements, such as brewer's yeast or lactic acid bacteria (Bifidus) found in yogurt. When absorbed, they provide beneficial health effects, such as improving digestion, fighting diarrhea or certain digestive system infections.

Probiotics are live microorganisms. They exist naturally in the digestive system of humans forming the intestinal flora or the intestinal microbiota, they have a beneficial effect on the body.

V.1.2. The microorganisms used for the production of P.O.U.

Generally, four types of microorganisms are used. These are yeasts and molds, micro-algae, and bacteria:

• The yeasts generally used as P.O.U are mainly : 

- Saccharomyces cerevisiae: used mainly as a food additive. 

- Candida utilis: after inactivation by heating, it is used as a nutritious food, since it is rich in proteins and free amino acids, and has a meaty flavor.

• The mold, Fusarium venenatum, is used in the production of Quorn, which is a brand of mycoprotein-based meat substitute with a taste resembling chicken meat.

• Cyanobacteria can be used as food supplements, for example spirulina, produced mainly from the species Arthrospira platensis. Spirulina is rich in proteins (60% protein) and vitamins, as well as mineral salts and trace elements.

• The bacterium Escherichia coli is widely used in the production of proteins because their genetic material is known. Other bacteria are used such as Bacillus subtilis, Streptomyces.

V.1.3. Criterion for choosing a microorganism for the production of P.O.U.

Before being used as a source of P.O.U, the microorganism must :

• Be non-pathogenic

• Have a high protein level.

• Have a high growth rate;

• Be easy to harvest;

• Have good resistance to variations in production conditions.

V.1.4. Example of industrial production of P.O.U.

 Yeast production:

Yeasts intended for cell production are grown in large aerated fermenters in molasses-based media which contain a large amount of sugars as a source of carbon and energy, as well as necessary minerals, vitamins and amino acids. to their growth. To complete the medium, a source of phosphorus (phosphoric acid DNA synthesis) and a source of nitrogen and sulfur (ammonium sulfate protein composition) are added. To achieve fermenter volumes ranging from 40,000 to 200,000 liters.

Several intermediate scaling steps are required.

➢ From the pure strain received from the laboratory, the yeast is first inoculated in tubes and flasks to prepare the inoculum, a biological source for industrial production.

➢ The inoculum is intended to be propagated in fermenters of increasing sizes. From the initial test tube to the final phase in a commercial fermenter.

➢ Each fermenter is seeded with yeast, with precise additions of molasses, nutrient salts and air, and strict temperature and pH controls to ensure the proper development and balance of the cell. Commercial fermentation lasts approximately 16 hours.

➢ At the end of growth, the culture medium is removed by filtration and the cells washed with water and recentrifuged, until they are light in color.

 Production of bacteria

Probiotics are generally lactic acid bacteria, Gram (+), which require rich culture media.

  On an industrial scale, the optimization of growth conditions represents an important step in the production process. First of all, the choice of components of the culture medium must meet the nutritional needs of the microorganisms, regulatory criteria and economic criteria.

The best growth conditions for producing e.g. L. acidophilus bacteria are 40 g/L glucose, 20 g/L peptone, 20 g/L yeast extracts, 5 g/L sodium acetate, 3 g/L of sodium citrate (pH=6.0 and T=30°C).

During industrial production, fermentation is carried out in closed culture (batch) or Fed-batch. However, continuous fermentation generally gives better yields but it creates risks of contamination and can lead to a loss of the characteristics of the strain over time.

The biomass is recovered by centrifugation. It comes in the form of “very concentrated bacterial paste”, which must then be dried with great care in order to keep as many bacteria as possible alive.

V.2. Main products of industrial microbiology :

Industrial microbiology makes it possible to produce numerous substances (agri-food, medical, pharmaceutical, agricultural, etc.). Some metabolites are synthesized and appear in the culture medium during the growth phase (primary metabolites), and others appear when the organism has completed the growth cycle and enters the stationary phase (secondary metabolites).

Microbial products are classified into 3 categories: primary metabolites, secondary metabolites and enzymes.

V.2.1. Primary metabolites :

They include compounds synthesized by microbial cells during the growth phase (trophophase).

A primary metabolite P1 is formed essentially when cells divide, during the logarithmic growth phase, called trophophase; the production curve and the cell population curve are almost parallel, with a slight deviation. Microorganisms do not produce P2 secondary metabolites until they have completed their growth phase and entered the stationary phase, called idiophase (Fig).

They include amino acids, nucleotides and end products of fermentations such as ethanol and organic acids.

Table: Some industrial products resulting from primary metabolism :

V.2.1.1. Production of amino acids :

Nowadays, amino acids constitute an important industrial product derived from microorganisms.

In nature, microorganisms rarely synthesize a quantity of amino acids exceeding their needs, therefore at an industrial level, to increase the production of the desired amino acids, mutants have been isolated and selected to characterize hyperproductive microbial strains (Ex: Corynebacterium glutamicum , Brevibacterium flavum, Escherichia coli).

To make amino acids, fermentation tanks are filled with molasses and sugar ingredients such as sugar cane, corn and cassava. Ideal conditions are achieved by agitation, oxygen supply, temperature and pH levels. The desired amino acids are then purified from this fermented broth.

Table: Examples of amino acid production by microorganisms :

Use of amino acids:

66% of the volume of amino acids produced is devoted to human nutrition. Glutamate is used in seasoning, glycine, cysteine and alanine are also used as additional factors. Mixtures of amino acids are used to improve the flavor of certain products in the food industry.

 Glutamic acid is used to produce monosodium glutamate, a flavor enhancer.

   Lysine is used as a dietary supplement in cereals.

Table: Industrially produced amino acids and microorganisms involved in their synthesis :

They are also used in medical applications (surgical treatments, liver diseases, ulcers).

V.2.1.2. Production of organic acids

An organic acid is a compound capable of releasing a cation (positively charged ion) H+ in aqueous media. The most common organic acids are: lactic acid, acetic acid, propionic acid, butyric acid, citric acid, etc. Organic acids are additives widely used in food preparation as pH balancing agents, preservatives, acidifiers, antioxidants, etc.

 Production of citric acid : Citric acid (C6H8O7) is a product of the metabolism of molds, and more particularly of Aspergillus niger (the best producing mushroom). The production of citric acid by this mold is mainly done in submerged culture, it is sensitive and limited to several factors such as iron, zinc, manganese and phosphate.

Also, the production of citric acid by Aspergillus niger is strongly influenced by the type and concentration of:

 The carbon source, mainly sucrose, must be between 140 and 220 g/L.

 The nitrogen source must not exceed 0.4 g/L.

 The use of ammonium salts, such as ammonium sulfate (NH4)2SO4, is preferred for the production of citric acid.

More than 60% of the annual production of citric acid is devoted to the food industry (drinks, jams, juices, etc.), it not only gives a tangy flavor to foods, it is used as an antioxidant and agent. pH balancer in food preparation and as an emulsifier in the manufacture of dairy products, it is also used in the composition of detergents and cosmetics.

 Production of lactic acids:

Industrial lactic acid is synthesized by Lactococcus delbrueckii grown at 50°C, and by Lactococcus bulgaricus at 44°C. Fermentation is anaerobic and lasts 6-7 days and the yield is 80-90g of lactic acid per 100g of carbohydrates.

Lactic fermentation is involved in the industrial production of several products:

 Fermented milks: yogurts, cheeses (Their beneficial contributions consist of improving the quality of fermented products)

 Fermented plant products: olives, soy milk

 Food preservation: inhibition of pathogenic or spoilage flora (probiotic and bacteriocin properties).

 Production of vitamins:

Vitamin B12 is produced only by micro-organisms (bacteria and archaea). Low concentrations of vitamin B12 are found in fungi or algae, which can be explained by the growth of bacteria on them. ¬face.

Several bacteria are commonly used for the production of vitamin B12, for example Bacillus megaterium, Pseudomonas denitrifi-cans or Propionibacterium freu¬den¬rei¬chii and Propionibacterium shermanii.

The culture medium used for the industrial production of this vitamin is essentially composed of :

 From a carbon source (Glucose or beet or citrus molasses).

 A source of nitrogen (fish meal, corn steep, casein hydrolyzate, etc.).

 A buffer (CaCO3).

 From a source of cobalt (CoCl2).

 From a precursor: 5', 6-dimethylbenzimidazol.

Fermentation takes place between 27°C and 30°C, for 3 days, with stirring and aeration. The vitamin is extracted either by thermal shock or by acidification (sodium sulphite). Under these conditions, vitamin B12 is released. A concentrate of this vitamin is prepared after filtration and drying (75 mg of B12 are obtained for one kg of medium used).

Many vitamins are produced by microorganisms, such as vitamin A, E, D, B1, B2.

 alcohol production :

Yeasts : Saccharomyces cerevisiae, is used industrially for alcoholic fermentation, it uses glucose as a carbon source, other species of yeasts can convert xylose into ethanol (Pichias tipitis, Candida, etc.).

Bacteria: Xylose is converted into ethanol by several species of thermophilic bacteria such as Thermoanaero bacterethanolicus, Clostridrium thermohydrosulfuricum or by modified strains of Bacillus stearothermophilus. Mesophilic bacteria such as Escherichia coli and Klebsiella oxytoca or K. planticola are also capable of fermenting pentoses.

 Production of Biogas by the methanization process :

Microorganisms have a major interest in the production of energy and in cleaning the environment of pollutants. Certain methanogenic bacteria are capable of producing methane, a natural gas widely used by humans as an energy source.

Biogas is a renewable energy produced by the anaerobic digestion of biodegradable organic materials of different categories (green waste, fermentable household waste, station sludge, livestock effluents and agricultural co-products, agricultural energy crops, etc.).

Biogas is a mixture containing mainly methane (CH4), the largest part (50 to 72%), carbon dioxide CO2 (25 to 50%), and other gases such as hydrogen sulfide, nitrogen, hydrogen and steam.

Depending on its methane purity, biogas could find numerous applications :

- Production of electricity and/or heat

- Green energy source

- Reduction in pollution from discharges

- Reduction of greenhouse gases.

- Production of an improved fertilizer

- Reduction of odors & pathogens Local industry

- Upgrading the image of farms

- Reduction of expenses

- Injection into the natural gas network

An archaeabacterium called Methanosarcina rumen found in the rumen of cattle and sheep, produces methane by digesting decomposing organic matter.

 Vaccine production :

A vaccine is a preparation of toxoids, inactivated or weakened microorganisms, or fragments of microorganisms whose objective is to protect the individuals to whom it is administered.

Anatoxin : Treated bacterial toxin, having lost its toxic properties but retained its immunizing properties.

Inactivated microorganisms : the microbe is whole, but in inactivated form, it is completely incapable of multiplying and therefore of causing disease.

Weakened microorganisms : This is the “attenuated” form, the microbe is less aggressive, it has been manipulated (for example it is incapable of multiplying).

The development and marketing of a vaccine is a long and complex process, manufacturing takes between 6 to 22 months and 70% of the production time of a vaccine is devoted to quality control to ensure that the batches of vaccines manufactured are strictly compliant.

Example of vaccines produced by microorganisms :

BCG against tuberculosis : This live attenuated vaccine is prepared from a strain of bovine tuberculosis bacillus, Mycobacterium bovis, which has lost its virulence on humans after cultivation on special media during numerous reproduction cycles.

The hepatitis B vaccine is produced using recombinant DNA technology. A plasmid containing the HBsAg gene (hepatitis B surface antigen) is inserted into classic baker's yeast, which then produces HBsAg. This is harvested and purified.

The anti-poliomyelitis vaccine is an inactivated vaccine, obtained from cultures of monkey kidney cells, isolated by trypsinization. The virus is inoculated into the confluent culture of cells and grows within three days. The culture medium is then concentrated and the virus purified.

V.2.2. Secondary metabolites :

  These are produced by many microorganisms, and have the following essential characteristics :

- They are not necessary for the growth of the organism.

- They have a wide variety of structures and biological activities.

- They derive through their own synthesis pathways, from intermediates or products of primary metabolism.

- Their synthesis takes place during the stationary phase of growth.

V.2.2.1. Antibiotics :

Antibiotics are natural organic molecules resulting from the secondary metabolism of filamentous fungi and bacteria, in particular actinomycetes. The latter produce them to eliminate the competing bacteria with which they compete in their biotope. The principle of action of antibiotics consists of selectively blocking a step of a mechanism essential to the survival or multiplication of bacteria. The mechanism targeted by the antibiotic is most often specific to bacteria and has no equivalent in eukaryotes and in particular in humans.

The industrial production of antibiotics is carried out in large fermenters, the size of which varies from less than 40,000 liters to more than 500,000 liters, and follows several conditions:

• The selection of strains of microorganisms that produce the most antibiotics.

• Determining the best growing conditions (temperature, nature of the support, etc.)

• The production medium must first ensure significant growth to lead to a high cell concentration at the time of production.

• The culture is very generally aerobic. Precursors can be added to the culture medium to direct the synthesis. This is the case of phenyl acetate for the production of ampicillin: the fungus producing Penicillin G will then produce Ampicillin.

• During the antibiotic production phase, cells use slowly catabolic sources of energy and carbon (lactose for example, for the production of penicillin; dextrin or starch for the production of macrolides).

• Fatty acids and their derivatives are often provided by oils in the form of triglycerides. The most used oils are: soybean, peanut, corn and rapeseed oils.

• Ammonium is the best source of nitrogen to ensure rapid growth. Ammonium salts are added to promote this phase while monitoring the concentration, to avoid the drop in production linked to too high a concentration.

V.2.2.2. Polysaccharides of microbial origin :

Polysaccharides are simple sugar polymers (monosaccharides), linked together by glycosidic bonds. They are formed by a single type of monosaccharide (homopolysaccharide), or are formed by several types of monosaccharides (heterogeneous heteropolysaccharide)

Industrial polysaccharides can have different origins, such as plants (cellulose, pectin, gum arabic and starch) and algae (agar-agar, alginate, carrageenan). Microorganisms, including bacteria and fungi, can synthesize industrial polysaccharides. Depending on their location in relation to the producing cell, there are three types of polysaccharides of microbial origin.

• Intracellular polysaccharides: these types of polysaccharides are difficult to extract;

• Wall polysaccharides: like chitin which exists in the wall of mushrooms, these types of polysaccharides are also difficult to extract.

• Extracellular polysaccharides: these types of extracellular polysaccharides do not form covalent bonds with the microbial cell wall. They are generally called exopolysaccharides (EPS), they are either secreted out of the microbial cell in the form of loose slime, or they coat the microbial cell in the form of a capsule called slime or ''capsular polysaccharides (CPS)'' .

Exo-polysaccharides of microbial origin fulfill several physiological functions for the microorganism:

• They can protect the microorganism against desiccation,

• They allow the microorganism to escape from the immune system,

• They act as a barrier against viruses and chemical agents,

• They facilitate the attachment of the microorganism to different surfaces,

• They are energy reserves.

Alginate: This polymer is biosynthesized by Gram-negative bacteria, Pseudomonas sp and Azotobacter vinlandii.

Alginate is applied in the food industry for their emulsifying, gelling, thickening and stabilizing properties, especially in milk-based products (ice cream, ice cream) as well as pastries. It can also be used to encapsulate medications, in the form of alginate beads, and for the manufacture of beauty products.

Dextran: Is a homopolymer synthesized by several microorganisms, including Leuconostoc mesenteroides, it is produced industrially from sucrose, by the enzyme dextranasesucrase. Dextran is used for plasma replacement and to measure blood lipoproteins. It is also used for the manufacture of artificial tears, in lacrimal insufficiency or for contact lens wearers.

Xanthan gum is a heteropolysaccharide obtained by the aerobic fermentation of sugars by bacteria of the genus Xanthomonas, including Xanthomonas campestris, Xanthomonas carotae, Xanthomonas malvacearum and Xanthomonas phaseoli.

It is used as a food additive under the code E415 for its thickening and gelling properties in order to modify the consistency of foods.

V.3. Microbial enzymes :

Enzymes are biological catalysts (biocatalysts) of a protein nature which, due to their specific properties, participate in the synthesis of several molecules. They are responsible for catalyzing and accelerating certain chemical reactions.

Microbial enzymes are obtained from different microorganisms such as bacteria: Bacillus subtilis, Bacillus licheniformis, and fungi: Aspergillus spp., Rhizopus (table). They are considered to be superior enzymes used particularly in industrial applications for the production of detergents, food and beverage products, paper and textiles.

Microbial enzymes offer several benefits to industries, such as high catalytic activity and specificity, increased stability, are non-toxic in nature and have more efficient production cost-effectiveness.

They are used in all fermentation industries, whether for:

• Agri-food products (fruit juice, sugar, condiments, additives, meat, cereals, milk, cheese, ready meals, bread, biscuits). Examples:

- Amylase: for the degradation of starch (glucose); acceleration of yeast activity for bread production; conversion of cereal starch used in syrup production (sweet taste); clarification of wine and beer.

- Cellulase and pectinase: clarification of fruit juices (degradation of fruit pectin in the production of fruit juices and wines).

• The pharmaceutical and cosmetic industry (antibiotics, vitamins, beauty cream application products, etc.).

• The biomedical industry (recombinant proteins, reagents, etc.).

• The biochemical cleaning and decontamination industries (laundry detergents, detergents, degradation of grease stains, water and surface treatment).

Example of enzyme-producing microorganisms :

The typical industrial process for enzyme production is deep aerobic culture using a microorganism that produces large quantities of an extracellular enzyme. Two microorganisms are mainly used: Bacillus and Aspergillus.

The enzyme production environments must be rich and complete, providing all the nutrients (source of carbon, nitrogen, phosphorus, trace elements, vitamins, etc.). In addition, it is necessary to add the substrates of the desired enzyme, for example: Starch for the production of α-amylase, or Cellulose for cellulase production.