by Lena VOROBYOVA, Dr. Sc. (Biol.) M.V. Lomonosov Moscow State University

Propionic-acid bacteria (PAB) happened to be the centerpiece on the Third International Symposium held at Zurich, Switzerland, in the summer of 2001. To begin with, participants in this forum conferred an international award on a Russian biologist-Professor Lena Ivanovna Vorobyova - for her signal contribution to the studies of propionic bacteria, which are a unique form of life. The author of the present article, Dr. Lena Vorobyova, looks into the problems of present-day microbiology.

Every living breath on earth depends on microorganisms for existence and subsistence, be it directly or indirectly. Microorganisms are the most active little things that divide the fastest and have the most intensive respiration rate. Their high catalytic activity is also manifest in chemical transformations of various organic substances and of such elements as bivalent iron, tellurium, sulfur, phosphorites, salts of selenium, among others. Any oxidizable cationic or anionic compound serves as a source of energy for bacteria, the lithotrophs, that fix carbon dioxide, or СО ₂ .

The diversity of microorganisms and substances they live on, their stupendous adaptability to changes in the environment - all this enabled the famous Louis Pasteur, the father of contemporary microbiology and immunology (this French chemist and bacteriologist was also an honorary member of the St. Petersburg Academy of Sciences) to make the prophetic conclusion: "Bacteria are going to have the final say."

So far over 100 thousand bacterial species have been described, or about 10 percent of the total number. Some of these are what we may call useful bacteria-like, for instance, lactic-acid, propionic(-acid) and bifidobacteria, or the probiotics, which are always present in the human intestine and on the skin; by inhibiting the growth of harmful, putrescent microflora, these organisms have a positive effect on human health.

As to propionic bacteria (PAB), they were "domesticated" by cheese-makers as far back as nine thousand years ago. Today they are used for the production of vitamin B ₁₂ (which is implicated in the synthesis of vital substances - methionine and nucleic acids, and is also involved in hemopoiesis, or blood forming).

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PAB are employed in the output of propionic acid, in bread-backing, and in medicine (including veterinary medicine).

THE "PORTRAIT" OF PROPIONIC BACTERIA

A skilled microbiologist will always tell apart PAB from other just as small bacteria (measuring 1.0-1.5 x 0.5 mkm): propionic bacteria are remarkable for what we call a palisade, or columnar, pattern of constituent cells, which resemble hieroglyphics, with a large number of dents and inflations visible with a scanning electron microscope. Besides, PAB do not form spores-they are motionless, facultative anaerobes, that is they can grow in the presence of a small amount of oxygen and in its absence alike.

Propionic-acid bacteria get their name from propionic acid they produce (alongside acetic acid and СО ₂ ). They belong to the genus Propionibacterium, which also takes in lactic, or classical, and skin bacteria. Now, lactic-acid bacteria are isolated from milk and hard rennet cheeses; skin bacteria are the only anaerobic microflora of healthy human skin and a component of substances contained in the rumen, the first stomach of a cud-chewing animal, such as a cow. Back in 1975 Bernard Halpern, a French microbiologist, demonstrated the stimulating effect of this microflora on the immune system and its inhibitory action on tumor growth. These conclusions were subsequently confirmed in works of other scientists.

Nature has endowed propionic bacteria with a range of synthetic capabilities accounting for the liability of their exchange and adaptability to various conditions. They can grow in a medium containing just glucose, mineral salts and two or three vitamins; they can fix molecular nitrogen and shift from the anaerobic to the aerobic mode of life; this transition if effected with the aid of vitamin В 13 synthesized by bacterial cells. In the absence of this vitamin (when, for instance, bacteria are cultivated in a medium not containing cobalt, а В 13 component), PAB change from the anaerobic to the aerobic type of existence.

PAB are getting energy through propionic-acid fermentation which is also taking place in mammalian cells, where, however, cyclic reactions are of reverse directionality and, as a consequence, lead to the degradation of some amino acids, fatty acids and thimine.

Up until 1954 PAB were cultivated only in anaerobic (oxygen-free) conditions. Many microbiologists would not think it possible to have propionic bacteria grow under aerobic conditions, that is in the presence of oxygen and other gases of the air. However, propionic bacteria, as it later turned out, have a short respiratory chain and are capable of oxidative phosphoryla-

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tion which implicates cytochromes* as electron carriers. But unlike higher organisms where molecular oxygen works as an oxygen acceptor, propionic bacteria also make use of nitrates and fumarates (with cytochrome b as carrier); their respiration is anaerobic, one that occurs only in bacteria.

Another important characteristic of PAB is intensive synthesis of vitamin B ₁₂ which is present in cells of native strains in amounts hundreds of times as high as what is necessary for the reaction of propionic-acid fermentation.

BOOSTING VITAMIN В ₁₂ PRODUCTION

For commercial purposes vitamin B ₁₂ is produced via microbiological synthesis involving PAB as the chief agents.

In the 1970s our scientists undertook the job of boosting the output of this vitamin and bringing down its costs. We saw what could be done about it. Routine methods of selection and assay of active strains were all too laborious and could take a very long time. What we needed was an essentially new rapid method. And we did develop this method just within a year. Here's what it is all about. A suspension of mutagen-treated cells and a suspension of control cells (not mutagen-treated) were mounted on a solid substrate containing radioactive cobalt ⁶⁰ Co. After 10 days of incubation under anaerobic conditions the mass of no less than 100 bacterial colonies was determined (using sterile substrates), and so was their radioactivity (using gamma-radiation counter).

* Cytochromes - respiratory enzymes responsible for electron transport in living cells from oxidized substances to electron acceptor. - Auth.

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Cycles of bacterial propionic-acid fermentation.

Our rapid assay method made it possible to inspect 400 to 500 bacterial colonies daily. Their selection was done in a graduated manner (with mutant bacteria selected). The bacterial strain we obtained exceeded the initial one tenfold in B ₁₂ productivity. We passed this strain to the chemical- pharmaceutical industry (L.Y. Karpov plant in Moscow).

"PROPIOVIT": NEW FODDER ADDITIVE

Vitamin В ₁₂ , if added to green fodder, increases its nutritional value for cattle and poultry and thus contributes to their better productivity Besides, propionic bacteria reduce the excessive acidity of silages, while propionic and acetic acids formed in the process of fermentation are well assimilated. Yet another advantage: bacteria produce a large quantity of vitamins B ₂ and В ₁₂ (vitamin В ₁₂ is absent in plants). Propionic bacteria, when used as ferment in silage-making, go to make high- grade dietetic feed for cattle.

The non-toxicity of PAB, among other useful effects, makes these microorganisms a good remedy in veterinary medicine, both for cure and for prevention. One such patent remedy is Propiovit obtained from cells of Propionibacterium acnes which are isolated from the cow's rumen. It supplies animals with vitamin B ₁₂ , makes up for the deficiency of vitamins В ₁ B ₂ , В ₃ and B ₆ , adds to weight increment and reduces the mortality rate by half. As shown by our practical experience, every rouble spent on this feed additive gives a return of 19 roubles.

A NEW RECIPE FOR CHEDDAR

Cheese-making is, in fact, the oldest biotechnology developed by humankind. Now, cheese is a food product obtained by coagulating the casein of milk and incorporating a large portion of fat. Propionic bacteria play an important part here. To make cheese we take milk, table salt (sodium chloride), rennet (a soluble ferment found in the fourth stomach of calves) and a culture of lactic acid-producing bacteria. By varying these components and treatment techniques, we can obtain hundreds of cheese brands.

PAB are very important in upgrading the quality of cheese. Propionic bacteria are added together with lactic bacteria (both are compatible physiologically); the latter are implicated in the acidification or milk and in the precipitation of casein. At first lactose (lactic sugar) converts to lactic acid which is fermented by propionic bacteria, whereby propionic and acetic acids,

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and СО ₂ are formed, giving spice and flavor to the product.

Propionic bacteria also split protein into amino acids and volatile substances, such as acetoin, diacetyl, dimethylsulfide and acetaldehyde, the substances adding flavor and pungency to the best makes of cheese. Such is the conventional technique of cheese-making and curing.

We have suggested certain improvements. Joining hands with microbiologists of the Institute of the Butter-Manufacturing and Cheese-Making Industry at Barnaul, we have obtained a dry multistrain ferment ofpropionic bacteria superior to the pure monoculture in gas and acid productivity. Such dry PAB cultures are added to milk without their prior activation. Their economic effect is obvious. Add to this the convenience of transportation. Today Russia is making the best brands of cheese with the use of the multistrain PAB ferment.

LONGER SHELF LIFE FOR EGG POWDER

Foreign manufactures obtain egg powder by using chemical grade enzymic preparations, namely glucose oxidase and catalase, so as to eliminate carbohydrates which lower the moisture resistance of the powder during storage. Such treatment, however, inevitably changes the product's natural color and organoleptic characteristics. In plain English, the powder's taste and quality is affected as a result. We have suggested a different, bacteriological method - again, by using propionic-acid bacteria. Cultivated for 24 hours in liquid egg albumin, they digest carbohydrates. Besides, they contribute other substances, such as propionic acid (preservative), vitamins of the group В and free amino acids. And they split egg fats which cause egg powder to go off during long upkeep. Upon fermentation of the albumin by our method, the number of foreign bacteria in the product decreases at least by half; the physicochemical

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characteristics of the egg product are likewise improved - in particular, we obtain a larger volume of stable foam. For instance, fruitcakes baked after prior fermentation of egg albumin by PAB acquire a range of palatable qualities, such as whiteness, good structure and high porosity. Something to make your mouth water.

BREAD-BAKING

At the Third International Symposium at Zurich, Switzerland, on the subject of Propionibacteria we presented materials showing that propionic bacteria are good additives to a wheat bread leaven which also contains baker's yeast and lactic-acid bacteria. PAB protect the baked bread against the potato disease caused by some bacilli. Besides, certain essential - what specialists call theological - properties of dough are improved as a result (viscosity, plasticity, elasticity); there is an even distribution of pores throughout the loaf. Owing to the proteolytic (protein-splitting) activity of PAB, bakery products are enriched with amino acids and vitamin B ₁₂ , while the volatile compounds secreted by PAB microorganisms add to the taste and flavor. Today our innovative technology of wheat bread leavens has found wide use in Russia and elsewhere.

In the course of our PAB studies we were in for ever new surprises. Researchers of the Microbiology Department (M.V. Lomonosov Moscow State University) found propionic bacteria to be producing large amounts of enzymes that render harmless the aggressive forms of oxygen (the superoxide radical - O ₂ , the hydroxyl radicals - OH ^- , OH ^x ) and hydrogen peroxide. PAB also produce other substances capable of minimizing the lethal effect of chemical mutagens, ultraviolet rays and radiation.

Up until now certain plants, sea animals and chemical compounds were thought to be the main source of antimutagens, i.e. agents lowering the level of induced and spontaneous mutations. But now we know: many of the bacterial strains we have studied (their cells, to be exact) and extracellular protein compounds released by PAB reduce by nearly nine-tenths the noxious effect of substances causing point (intragene) mutations that can give rise to defective proteins and even arrest protein synthesis.

The antimutagen characteristics of propionic bacteria open up new spectacular vistas of their practical application. As a preventive remedy, for one, in food and feed additives.

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