Biotechnology - Yu.O. Sazykin 2006

Applied Biotechnology
Pharmaceutical products obtained via biotechnological methods in the pharmaceutical industry
Probiotics

Probiotics are living organisms and/or substances of microbial or other origin that, when administered naturally, exert beneficial effects on the physiological Functions, as well as the biochemical and behavioral Responses of the host Organism, thereby optimizing its microbiological status.

A symbiont is a participant in Symbiosis — the cohabitation of organisms of different species within the same ecological niche. The symbiosis of multicellular and unicellular organisms is one of the oldest phenomena in evolution, having emerged right at the inception of multicellular life. According to scientific consensus, life on Earth originated about 3–4 billion years ago due to natural causes, with microorganisms being the initial living forms. Some data suggest their evolution lasted for about a billion years. It was during this period that the entire diversity of biochemical reactions underlying the central METABOLISM of all living beings was shaped.

Multicellular Organisms evolved from unicellular ones in the global ocean (within the same ecological niche), and the symbiosis between them has been observed since their very origin. Throughout evolution, multicellular organisms developed mechanisms to regulate the Variability of their internal environment and established specific types of interaction with unicellular organisms. Currently, the following forms of symbiosis are recognized:

✵ mutualism — a mutually beneficial symbiosis;

✵ parasitism — one partner derives unilateral benefit at the expense of the other;

✵ commensalism — one partner gains a unilateral benefit without causing any harm to the other organism;

✵ neutralism — partners do not exert any noticeable influence on one another.

All these forms can be observed when examining the symbiosis between unicellular microorganisms and multicellular hosts. When speaking specifically of human-microbial symbiosis, it refers to the fact that the Skin, mucous membranes, and Body Cavities communicating with the external environment are colonized by a vast population of symbiotic microorganisms.

Examples include the gastrointestinal tract (GIT) — from the Oral Cavity to the rectum; the respiratory tract — from the nasopharynx to the alveoli; and the Large Intestine.

Microorganisms are particularly abundant in the large intestine, which harbors up to 20 genera and over 100 species of microbes exhibiting immense metabolic activity. Due to this activity, the microflora of the large intestine is sometimes compared to the Liver, which boasts the highest metabolic rate in the body. Naturally, these gut microorganisms significantly influence human physiology (Fig. 18). The large intestinal microflora participates in Digestion: Proteins, CARBOHYDRATES, and other dietary components entering the gut are broken down by Enzymes produced by symbiotic microorganisms. They are also capable of producing cellulases, a complex of enzymes that digests Cellulose. While this function plays a minor role in humans, for certain herbivores and ruminants, the assimilation of cellulose relies entirely on symbiotic microorganisms. Furthermore, large intestinal microbes can carry out the Hydrolysis (facilitating the assimilation) of other dietary components. Specifically, lactic acid Bacteria break down lactose into lactic acid. Symbiotic microorganisms can also break down various Other Compounds ingested with food. For instance, they can reduce nitrates to nitrites and subsequently catalyze them. Since nitrites may exhibit mutagenic and carcinogenic (oncogenic) properties, neutralizing them allows microorganisms to prevent The impact of these potentially active oncogenic factors on The Human Body. However, it should be noted that certain symbiotic microorganisms can activate specific procarcinogenic substances.

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Fig. 18. Functions of symbiotic microflora

Symbiotic microorganisms supply the human body with certain nutrients, such as Vitamins (vitamin K, B vitamins, and to a lesser extent, carotenoids and other Fat-soluble vitamins). In addition to vitamins, they can produce organic acids (lactic, acetic, succinic, and formic), which are absorbed and utilized by the macroorganism. Some of these microorganisms can synthesize Essential Amino Acids, notably Lysine, Tryptophan, and Threonine. At the same time, symbiotic microbes facilitate the utilization of Trace Elements by forming complexes that are more efficiently absorbed by the intestinal walls, which is especially important when Dietary intake of these trace elements is insufficient.

Symbiotic microflora also acts as a stimulator of nonspecific Immunity (an antigenic stimulus) and ensures the nonspecific resistance of the macroorganism.

Finally, having colonized the GIT (forming the resident microflora), symbiotic microorganisms prevent the establishment of foreign microbes, including pathogens. The environment contains a multitude of microorganisms that would otherwise find favorable conditions for growth in the human gut, given its Abundance of nutrients, constant Temperature, and high humidity. Yet, pathogenic microorganisms (such as putrefactive bacteria and producers of various toxins and toxic compounds, e.g., Clostridium botulinum) do not proliferate in the human intestine. Why does this not happen? Because the ecological niche is already occupied by symbiotic microorganisms that obstruct the colonization of foreign microflora.

The functions of symbiotic microflora were largely uncovered over the course of the past century, with experiments on germ-free animals (gnotobiotics) playing a pivotal role. Gnotobiology is a branch of biology focused on studying germ-free animals into which specific microorganisms are introduced, allowing researchers to observe how microflora affects the vital activity of animals and birds. For instance, to obtain a germ-free chick, a chicken egg is thoroughly treated with antiseptics to destroy all microorganisms On the surface and within the shell pores, and then placed in an incubator. After 21 days, a chick hatches and is transferred to a sterile chamber supplied with sterile air, where it is fed sterile food and given sterile Water.

Germ-free animals can also be obtained via Cesarean Section; for example, a pregnant sow undergoes a C-section, after which half of the piglets are placed in sterile conditions and the other half in conventional conditions for comparison. Based on such experiments, it was established that:

✵ germ-free animals live somewhat longer than conventional ones. At the beginning of the last century, I. I. Mechnikov hypothesized that one of the causes of Aging is the poisoning of the human body by putrefactive microflora inhabiting the large intestine. Mechnikov's idea that putrefactive microorganisms could be combated by introducing other microbes that suppress their growth was further developed, ultimately serving as the basis for the creation of probiotic preparations;

✵ these animals require large amounts of nutrients, vitamins, and organic acids, and are less efficient at assimilating certain Organic compounds;

✵ they are extremely sensitive to any weakly virulent or even opportunistic infections. When opportunistic bacteria enter a sterile environment, they immediately colonize the GIT and, encountering no competition, penetrate the bloodstream, causing infection and rapid death of the animal. This is attributed to the absence of nonspecific immune responses in such animals, which are normally induced by symbiotic microflora.

What specific symbiotic microorganisms inhabit the GIT? Their Classification is constantly evolving, so let us examine the main groups present in the digestive tract.

The first group comprises bifidobacteria (Bifidobacterium bifidum, longum, infantis, breve) and lactic acid bacteria (Lactobacillus acidophilus, plantarum), which are Gram-positive, anaerobic microorganisms (strict anaerobes), typically devoid of catalase and non-spore-forming. This is the most numerous group of symbiotic microorganisms. During metabolism, they produce lactic acid (sometimes exclusively) along with other organic acids (such as acetic acid, etc.).

In the intestinal contents of a breastfed infant, bifidobacteria account for up to 99% of all microbial populations. Their titer reaches 10¹¹ — 10¹² per 1 g of intestinal contents, forming the creamy mass that characterizes the excrement of a breastfed baby. As complementary foods are introduced, other microorganisms join the infant's gut microbiota. Among them, lactobacilli, or lactic acid bacteria, play a crucial role. The count of lactobacilli in healthy individuals ranges from (5 — 50) ∙ 10⁹ per 1 g of intestinal contents.

Most of the aforementioned bacteria are entirely harmless microorganisms that under no circumstances cause pathological processes. However, There is a species called Lactobacillus plantarum (found in plant juices on plant cuttings), which is also present in the human intestine. This microorganism does not belong to the category of entirely harmless lactobacilli. In individuals with Various Forms of immunodeficiency (AIDS, autoimmune diseases, etc.), Lactobacillus plantarum has even been detected on Heart Valves in cases of endocarditis.

Another genus of intestinal lactic acid bacteria is enterococci, which were relatively recently segregated from the genus Streptococcus. There are two major species of enterococci: Enterococcus faecium and Enterococcus faecalis. Among strains of the latter species, cultures capable of triggering pathological processes are frequently encountered.

The second group of bacteria comprises opportunistic and putrefactive bacteria (essentially, these are the same microorganisms, and this classification is somewhat arbitrary). As their name implies, opportunistic bacteria can trigger pathological processes under specific conditions. They permanently inhabit the human body and cause infections either when the host is weakened or when the population of opportunistic bacteria surges significantly. In the latter case, they induce low-grade inflammatory processes in the gastrointestinal tract (such as enterocolitis and dysentery-like conditions). Furthermore, they may colonize atypical sites, including the pancreatic and Bile ducts, as well as the Gallbladder itself, instigating pathological processes there (under normal conditions, these areas are sterile and devoid of symbiotic microorganisms).

Finally, in immunocompromised individuals, opportunistic microorganisms can enter the bloodstream and cause focal inflammatory conditions (endocarditis, Arthritis, pustular diseases, and even Sepsis). The most significant group by medical importance (rather than sheer numbers) is the Enterobacteriaceae family, commonly known as the enteric group of microorganisms. It includes both overt pathogens—such as certain Salmonella species responsible for typhoid fever, paratyphoid fever (Salmonella typhi and Salmonella paratyphi), and shigellosis (Shigella)—and a vast number of opportunistic bacteria. The latter category encompasses Escherichia coli, Proteus, Salmonella, Staphylococcus, and others.

The second most abundant genus of intestinal inhabitants (after bifidobacteria) is Bacteroides. These are Gram-negative, strictly anaerobic microorganisms. Their titer in one gram of intestinal contents reaches 10¹⁰–10¹¹. This group includes mucolytic strains that perform beneficial functions, although in weakened individuals, these strains may provoke pathological processes.

The next group is the genus Clostridium. These microorganisms perform a vital extralytic function (it is clostridia that produce extralytic enzymes); simultaneously, under certain conditions, they can cause pathological processes, for instance, upon entering the Abdominal cavity. The clostridia also include several notable pathogens (the causative agents of gas gangrene, tetanus, and botulism).

Yeasts of the genus Candida are found in the intestine (as well as other body cavities) as opportunistic microorganisms that can cause thrush when overproliferating. The administration of broad-spectrum Antibiotics eradicates the resident intestinal microflora; since these antibiotics do not affect Candida yeasts, the absence of competition naturally leads to the overgrowth of these microorganisms.

Thus, the paramount role among these two Major Groups of microorganisms belongs to bifidobacteria and lactic acid bacteria, which suppress The activity of opportunistic bacteria through their antagonistic action. In doing so, they exert complete control over the population size of opportunistic and putrefactive bacteria, maintaining it at a level that is safe for human physiology.

Symbiotic microflora is divided into mucosal (attached) and luminal microflora. Mucosal bacteria proliferate on The surface of the epithelium, whereas luminal bacteria attach to solid food residues, as many bacterial species require attachment to a solid substrate in order to multiply (divide).

The luminal group includes enterobacteria and certain strains of bifidobacteria and lactobacilli.

The antagonistic activity of bifidobacteria and lactic acid bacteria against opportunistic microflora (exhibiting bacteriostatic and bactericidal effects) manifests through several suppression factors:

✵ The production of lactic acid and other organic acids (such as formic acid), which are toxic to opportunistic and putrefactive microorganisms. Concurrently, these acids lower the pH of the intestinal contents (acidifying them), which also adversely affects the viability of the latter;

✵ the generation of peroxide compounds, The breakdown of which releases atomic oxygen that inhibits the Cells of the opportunistic microflora;

✵ the synthesis of antibiotic-like substances (bacteriocins, such as acidophilin and colicins) that suppress the metabolism of opportunistic and putrefactive bacteria by interacting with their Cell membranes and inducing cell lysis;

✵ The ability to attach to the intestinal epithelium, forming a dense biofilm on its surface through intensive proliferation, which acts as a barrier against the fixation of pathogenic microorganisms;

✵ the alteration of the Redox Potential of the environment, creating unfavorable conditions for the multiplication of opportunistic and putrefactive microorganisms.

The combined effect of these factors generally suppresses the excessive growth of opportunistic and putrefactive microflora. The state of the large intestine's microecological system in which lactic acid bacteria outnumber opportunistic and putrefactive bacteria is referred to as eubiosis or eubacteriosis. Conversely, the condition in which the population of bifidobacteria and lactic acid bacteria declines while that of putrefactive and opportunistic bacteria correspondingly rises is called dysbiosis. The overproliferation of putrefactive microorganisms leads to host intoxication, triggering a vicious cycle wherein secondary infections emerge and develop in a weakened individual. Furthermore, opportunistic microorganisms are capable of independently driving The Development of dysbiosis.

What causes dysbiosis to develop? One primary factor is the Introduction into the human body of substances with antibacterial activity. Among such substances, antibiotics take first place. Certainly, antibiotics have played—and continue to play—a colossal role in treating patients with various infections and saving hundreds of millions of lives, but they carry a negative side effect: they do not discriminate between mutualistic strains (lactic acid bacteria), pathogens, and opportunistic microorganisms. Broad-spectrum antibiotics suppress the entire gastrointestinal microflora, including lactic acid bacteria. Their suppression results in opportunistic and putrefactive microorganisms dominating over lactic acid bacteria once Treatment concludes. This occurs because the cells of putrefactive and opportunistic bacteria more frequently harbor Plasmids that confer resistance to specific antibiotics, which can be transferred from Cell to Cell. As a result, the population of opportunistic and putrefactive microorganisms becomes resistant to the action of various antibiotics.

In addition to antibiotics, human beings ingest substances via food that are deliberately added to prevent spoilage by putrefactive microorganisms. These are known as preservatives, which possess intrinsic antibacterial activity. Food may also contain antibiotics used in livestock and poultry farming to treat sick animals and birds, as well as to stimulate their GROWTH AND DEVELOPMENT. Food (especially of plant origin) may contain herbicides and pesticides that exhibit antibacterial activity.

The development of dysbiosis is facilitated by shifts in the host's hormonal status (during Puberty, Pregnancy, or menopause). The physiological state of the host influences the viability, reproduction, and Maintenance of the symbiotic (particularly mucosal) microflora. With aging, The amount of mucosal microflora (lactic acid bacteria) decreases, accompanied by shifts in hormonal status. This can lead to the rapid shedding of the mucosal microflora, leaving the epithelium exposed and vulnerable to colonization by opportunistic and putrefactive microorganisms.

Research shows that if an individual experiences severe stress, diarrhea may manifest the very next day, a phenomenon linked to stress-induced hormonal changes. If this stress becomes chronic, it thereby establishes the conditions conducive to the development of dysbiosis.

Probiotic preparations are designed for the Prevention and treatment of dysbiosis. In Russia, predominantly monovalent preparations are produced: Bifidumbacterin (based on Bifidobacterium bifidum), Apilac (based on Lactobacillus acidophilus), Colibacterin (based on Escherichia coli strain M-17), and Lactobacterin (based on Lactobacillus plantarum and fermentum). Combination preparations include, for example, Bificol (containing Bifidobacterium and E. coli M-17). Abroad, polyvalent (multistrain) preparations are common: Symbioflor (Enterococcus faecium + E. coli), Bificor (Bifidobacterium longum + Enterococcus faecium), Primadophilus (containing a complex of symbiotic microorganisms), and others, such as Enterol (based on yeasts) and Gastropharm (Lactobacillus bulgaricus, which is not a symbiont and therefore acts only temporarily). There are also preparations containing live cells of symbiotic microorganisms that are not strictly classified as probiotics, such as Bactisubtil (based on a culture of Bacillus cereus, mutant strain IP 5832).

Probiotics are particularly widely used in pediatric practice (for acute dysentery, salmonellosis, escherichiosis, viral diarrhea, diathesis, etc.). It is well established that the gastrointestinal microecological system is not yet fully formed in infants and children, making them prone to dyspepsia and diarrhea. In such cases, the administration of probiotics proves highly effective. Studies on the long-term use of probiotics in children's groups have demonstrated that children who systematically receive these preparations and fermented dairy products containing probiotic strains fall ill less frequently—not only with intestinal infections but also with acute respiratory infections—grow and develop better, and tolerate childhood infections more easily. Probiotic preparations are also utilized in the comprehensive treatment of intestinal infections (colitis, bacterial colpitis caused by staphylococci and E. coli, and enterocolitis associated with microfloral disruption featuring a deficiency or complete absence of bifidoflora).

Following the completion of antibiotic therapy, it is imperative to administer probiotics to restore normal microflora. Probiotics are also prescribed to weakened individuals suffering from somatic illnesses. A vicious cycle frequently arises where the weakened state itself causes dysbiosis, which in turn exacerbates the course of the somatic disease. Probiotic preparations are likewise prescribed to elderly individuals, as shifts in their hormonal status lead to a diminished count of bifidobacteria and lactobacilli. Therefore, to normalize the microflora, one must periodically (and constantly in old age) consume dietary food products alongside probiotic preparations. Examples include acidophilus milk, produced using an acidophilus starter culture, and Bifidumbacterin.

What is the technological process for obtaining such preparations? First, it is necessary to isolate strains of symbiotic microorganisms. These are harvested from the intestinal contents of healthy children and adults; for instance, the strain Enterococcus faecium was isolated from a child's intestine. Second, the isolated strains must be accurately identified. This is crucial because only strains of specific microbial species are authorized for the production of probiotics. These strains must possess the following properties:

✵ effective antagonistic activity, driven in turn by the synthesis of organic acids, which the strain must actively produce. When isolating bifidobacterial strains, formic and lactic acids serve as important antagonistic metabolic products. It is also desirable that these strains produce peroxides and antibiotic-like substances;

✵ an effective ability to attach to the intestinal epithelium, in the case of mucosal-associated microorganisms;

✵ not hydrolyze intestinal mucus, which has a protective effect. Certain microorganisms produce mucopolysaccharides that degrade (hydrolyze) this mucus;

✵ not damage intestinal epithelial cells (cholecytes).

Selected strains are invariably tested for pathogenicity

and toxicity in vitro on cholecyte cell cultures and in susceptible animals. Probiotics must not exhibit side effects even when administered in excessive doses. For safety verification, the preparation is introduced into susceptible animals in large quantities, sometimes up to several grams per kilogram of body weight.

Strains must be technologically viable, meaning they must grow and multiply efficiently on artificial nutrient media. They are processed via lyophilization: frozen to low temperatures and subsequently dried under low pressure. Accordingly, the selected strains must be cryoresistant and withstand the drying Procedure.

Strains meeting all these requirements are sent to a control institute, from where they are transferred to pharmaceutical production facilities accompanied by documentation reflecting their characteristics. In factory laboratories, the strains are plated onto artificial nutrient media and verified for compliance with passport data (genus, species, biological properties). Only then are they utilized for the production of probiotic preparations. Under industrial manufacturing conditions, these strains are plated out to obtain isolated colonies, which are subsequently subcultured into Agar or liquid nutrient media (for example, lactic acid bacteria thrive in skim milk).

Bifidobacteria, lactobacilli, and enterococci are all auxotrophs; that is, for their growth, they require a range of nutrients and trace elements and cannot independently synthesize amino acids, purine and pyrimidine bases, or vitamins, which must therefore be provided by the nutrient medium. Raw Materials approved for use in the food industry are employed for the cultivation of these bacteria, since the resulting preparation grown on these media is subsequently intended for internal administration.

Milk protein (casein), enzymatically hydrolyzed using Trypsin and Pepsin to yield tryptone or peptone, typically serves as the source of amino acids. Yeast extract, obtained from yeasts of the genus Saccharomyces (brewer's or baker's yeast), is utilized as a source of vitamins as well as pyrimidine and purine bases. Salts of magnesium, manganese, and zinc are added to the nutrient medium as trace elements for the cultivation of lactic acid bacteria. Lactose or glucose generally serves as the energy source.

Typically, lactic acid bacteria are cultivated for 8 to 16 hours (a relatively short Fermentation period). Strains are harvested at a growth phase that ensures maximum cell survival, which in turn guarantees extended shelf life during subsequent product manufacturing. All these parameters are determined empirically and often constitute the manufacturing company's know-how. As a rule, the end of the logarithmic phase or the beginning of the stationary phase is selected, depending on the specific culture (strain). Upon completion of the cultivation process, a bacterial suspension is obtained containing 10⁹ or more cells per ml. These cells are harvested using continuous-flow centrifuges or separators, yielding a cream-colored, sour-cream-like mass with a specific sour milk odor (resembling compressed yeast). A solution of cryoprotectants (protein-based substances such as skim milk or gelatin; carbohydrates such as lactose or sucrose) is added to the flowing biomass to yield a dense cell suspension, which is then dispensed into ampoules. Subsequently, these are frozen in liquid nitrogen and subjected to lyophilization. The dried mass acquires a vesicular appearance, is pulverized, and its titer is determined, after which it is dispensed into Glass or plastic vials or blended with a culture of another strain. In the latter case, preparations containing multiple symbiotic microorganism species are obtained.

Available formulations include vials (Bifidumbacterin), ampoules (Bificol, containing bifidobacteria and Escherichia coli), capsules (the Indian preparation Nutrolin-B, containing lactobacillus strains), or sachets (Bifidumbacterin manufactured by Partner, Russia).