The Advanced Probiotic
Dr. Dilip Kumar, MD
Pharmed Medicare (P) Ltd.

Introduction

Archives of Lactic acid bacilli from India

India, one of the oldest cradles of ancient wisdom, has been a contributing to the wisdom of mankind immensely. Charaka Samhita a treatise (supposed to be written in 1,000 B.C.) On ayurvedic medicine speaks of “Jataragni”(fire in the stomach or in the larger sense, Metabolism in the G.I.T.) as the sustaining force of all living beings. one of the first line treatment for the morbidity or disease of this process is “Takra” or fermented milk. He goes to the extent of calling the fermented milk “Amrita” or elixir. He speaks vividly about its utility is not only disease related to G.I.T., but also other disease like “Grahani” (Malabsorption syndrome), “Rajayakshma” or Phthisis, “Raktapitta” or (bleeding disorders), “Arshas” (haemmorrhoids or Piles), “Prameha” (hyperureas including diabetics mellitus). This gives the varied, but established applications of Lactobacilli in Indian subcontinent.

The concept was rediscovered in the west, and found popular acceptance. The doctrines of Ayurveda, which considers “Jataragni” or the metabolism of G.I.T as the cause of all pathologies is retold in the theory of “Autointoxication”.

The fermented milk consists of many useful bacteria, which in fact help digestion of the recipient, by producing various digestive enzymes & other metabolism enhances. In addition they help formation many immunoglobins as well as antibiotic like bacteriocins, which act as bactericidal on pathogenic bacteria, a measure is known as “biological control” in the ecological parlance.

Consuming bacteria instead a drug that kills bacteria for an infection was an entirely new concept to the inventors. Hence they called these microbes as “Probiotics” that means “for life” in Greek. These actually increase the number of beneficial bacteria in the gut at the cost of pathogens, hence get their apt name. But, they also get destroyed by the factors which affect the delicate intestinal environment like, stress, aging, antibiotics, changes in food habits (contaminated food)

Dr. Elie Metchinkoff, a Russian Scientist who propounded the “theory of longevity” has spoken about increasing one’s longevity by consuming yogurt rich in Lactobacillus. bulgaricus and Staphylococcus This discovery was thought to mitigate intestinal pathology & morbidities due to aging process.These strains failed partially because these species are not generally found in the intestinal microflora, hence were unable to cross the acid barrier or colonize in the intestine.


In the Unites States acidophilus milk was used for the treatment of diarrhea and constipation. When the tablets of Lactobacillus acidophilus were developed, the concept and product eventually were discarded, as the tablets did neither have considerable amounts of viable cells nor they produced any clinically significant effect.

The research for viable probiotic that can take up the pharmaceutical, storage & gastric acid challenges, as well as colonize in the intestinal flora. This led to the discovery of Lactobacillus Sporogenes in 1933 by L.M Horowity-Wlassowa and N.W. Nowotel now, which was published in “Bergeys Manual of determinative bacteriology”. Due to the simplification of taxonomical Cataloging and Characterization like “Spore bearing rods which produce lactic acid, are faculative or aerobic and catalase positive, have generally and correctly assigned to genus bacillus”, hence Lactobacillus Sporogenes was transferred to Bacillus coagulans.

Lactobacillus sporogenes was isolated in 1949, by Nakayama of Yamanashi University Japan, which was renamed based on its morphological physiological character as Bacillus coagulans.Hammer .

A meta analysis of 200 papers on L..acidophilus use in diarrhea found that only very few had controlled studies, which were capable of demonstrating an antidiarrheal effect in 1975. The later studies suggested poor activities in the previous studies.

S.thermophilus and Lactobacillus bulgaricus influence the presence of L.acidophicus and other enterobacteria to proliferate. Lactobacillus acidophilus and other enterobacteria to proliferate. Lactobacillus acidophilus has been a subject of three human studies on Candida of G.I. tract. Although they showed positive results, the samples were small & were not a part of co-ordinated research. The L.acidophilus yogurt is believed to be the cause of longevity in some Eastern Europeans, who consumed it as a staple food traditionally.

Lactobacillus is the most extensively studied and documented strain in the world. This strain stabilizes the intestinal flora, destroys pathogenic strains. Its ability to relieve gastrointestinal disorders and bacteria and viral infections are extremely well documented, as it is capable of anti-viral and anti-bacterial activities. It is capable of producing many immunoglobulins, which strengthen the immune system. There are interesting studies going on about the capacity of Bacillus sporogenes to reduce Cardiac risk, Cancer and HIV.


Antibiotics

Antibiotics are the pharmaceutical extracts, which are meant to exert a bactericidal action. They fail to recognize the pathogens and the probiotic bacteria and kill one and all. This leads to severe imbalance in the ecology if microflora, which eventually leads to G.I disturbance Lactobacillus acidophilus, was tried with antibiotics in 1950’s to retard or reverse the G.I. side effects of Antibiotics.

Ideal characters of probiotics

Every species has its own marked characters, which makes the selection of the right strain, more competitive. The clinical trails of different probiotics has yielded a mixed results, the main reason attributable to the inconsistent result may be selection of strain. This proves that selection of the strain for a condition is very important.

The ideal probiotic can be the one, which has the following characters

• Should be viable through pharmaceutical procedure bile and acid stable and stable in the storage conditions.
• Capable of attaching firmly to G.I mucosa (shouldn’t get swept by peristalsis)
• Should be able to inhibit or antagonize the gut microflora, so that it can survive the gut microbial competition.
• Should produce metabolites, which are friendly to the host.
• Should be antagonistic to G.I. pathogens
• Should produce anti allergens as well as anti-inflammatory factors.

• Anti infective due to the bacteriocins they produce along with the lactic acid.
• Controlled intestinal permeability, they help absorption of some nutrients which they help reduction of absorption of some nutrients ( e.g. LDL)

• Through their enzymatic activities. The strains of pro
• Lactobacillus GG
• Identified in 1987, through an invitro screening, this is the first bacterium colonize the human GIT.
• It has shown to protect immuno-compromised mice from Candida infection
• It has potential in primary prevention of atopic diseases.
• It is useful in pouchitis which is a complication of ileal reservoir surgery for crohn’s disease.

• autochtonus microflora, which are indigenous. They proliferate from the moment of birth and become a stable flora after the weaning period, and
• allochtonus microflora, which are microorganisms, introduced via ingestion and are therefore transient.

• Overgrowth of undesirable bacteria
• Overgrowth of yeast, including candida
• Parasites, such as amoebas
• Viral illnesses, such as measles
• Bad water or poor hygiene
• Too much sweets or starchy foods
• Too much alcoholic beverages
• Food allergies
• Certain medical drugs
• Frequent use of antibiotics
• Exposure to radiation
• Surgical complications
• Physical injury
• Excessive stress
• Environmental toxins
• Genetic sensitivities to any of the above
• The Gut Mucosal Barrier

Gut microflora play an important role in the intestine’s defense barrier. In the absence of a healthy intestinal microflora, antigen transport is increased, leading to allergies and increased allergic sensitivity.


Most antigens in the diet are excluded from the body by a well-functioning intestinal mucosal barrier. However, a fraction of these can antigens bypass this barrier in even the healthiest of people. The antigens are absorbed across the epithelial layer by a process called transcytosis. Transcytosis operates along two pathways. The main, or degradative, pathway involves lysosomal processing of the proteins into smaller peptide fragments. This reduces the immunogenicity of the proteins, thereby diminishing the antigen load. This is important in maintaining the host-defense system. Another pathway (paracellular leakage) allows for the transport of intact proteins, a process that can result in antigen-specific immune responses. Paracellular leakage of macromolecules does not occur in a healthy gastrointestinal tract because of intact, tight intracellular junctions that maintain the macromolecular barrier. Consequently, in healthy people, antigen transfer is well controlled, and aberrant antigen absorption does not occur.


Lactobacillus sp.: The enzyme mediated metabolism enhancers.
The microflora of the intestines play a pivotal role in the gut enzymatic system. The organism invariably depends on this microflora for the metabolism of various nutrients. Digests the proteins, fats and other nutrients in to easily assimiable components.

The role of Lactobacilli in nutrient metabolism:

Protein metabolism



Proteinases from Lactobacilli

Proteins+ H2O-------------------------------------->Polypeptides

Polypeptidases from Lactobacilli

Polypeptides + H2O -------------------------------------->Amino acids

The process of hydrolysis by the sporulated Lactobacilli is very slow on comparison with the rate of hydrolysis by the putrefactive microbes. The action of Bacillus coagulans on the protein substrate helps digestion and breaking down of proteins. Hence the ingested protein gets digested easily. This property is of great value in the field of geriatrics, convalescent and pediatric nutrition.


Fat metabolism
Lipases from Lactobacilli

Triglycerides (Fats)-------------------------------------->Fatty acids + Glycerol

The large molecular components of fat are broken to simpler easily digestible substrate. This very useful in cases where fat metabolism is impaired like stearrorhea, hypercholesterolemia, Diabetes. They also help deconjugate bile salts. These effects find application in pediatrics, convalescence and geriatrics.


Lactose metabolism

Lactase from Lactobacilli

C12H22O11 (Lactose)------------------------>C6H6O6Glucose)+ C6H6O6 (Galactose) +H2O

Glycolytic Systems of enzymes from Lactobacilli

C6H12 O6 (Glucose)-------------------------------------->Pyruvic acid

Lactic dehydrogenase enzymes from Lactobacilli

Pyruvic acid -------------------------------------------------------------->Lactic acid

Lactase, Glycolytic Systems of enzymes like b-galactosidase, Lactic dehydrogenase can be particularly of great practical value, as this is what a patient's system would lack in case of lactose intolerance.

Table 2.1 Antagonistic activities caused by lactic acid bacteria.

The lactic acid produced by lactic acid bacilli is of three types (Optical isomers of lactic acid), which are structurally of different configuration.

• L (+) lactic acid (Dextro rotatory)
• D (-)lactic acid (Levo rotatory)
• DL lactic acid (Racemic).
  

• Indigenous beneficial symbiotic bacteria.
• Potentially pathogenic bacteria

The homeostatic balance of these bacteria is found in the healthy subject. This is referred to as eubiosis, as this is required for the proper digestion, efficient absorption of nutrients.


Factors affecting eubiosis
view of the pressures of modern existence, the maintenance of a normal healthy, balanced microbial population (eubiosis) in gastrointestinal tract is a difficult task. Humans are often subjected to various stress conditions such as sudden changes in food consumption patterns, weather fluctuations, Pathological conditions which need antibiotics and/or immunosuppressive drugs and travelling long distances and too often. Under such adverse circumstances, the pathologic bacteria may supercede the gut environment to cause conditions like malabsorption, diarrhea, constipation, flatulence and such other GI conditions.

The presence of LAB in the human intestine is vital for normal human metabolism and for immunological activity. A reduction in their presence, whatever the cause, would lead to both gastrointestinal diseases and reduction of the host defenses. The ability of these bacteria to maintain a healthy ecosystem in the gut is referred as ‘probiosis’ or 'eubiosis', therefore LAB belong to the probiotic class and are able to produce a number of substances which hinder the growth of pathogenic bacteria: this process is called "antibiosis". LAB antibiosis is of considerable importance in prophylactic and therapeutic treatment of intestinal infections, because the presence of Lactobacilli renders the colonization of pathogens more difficult.

Antibiosis is related to the production of acidic compounds (e.g. acetate-lactate) which lower the intestinal pH and hinder the growth of pathogenic bacteria that usually require neutral or basic conditions.

LAB also produce different antibiotics, usually referred as "antibiocins" which are very effective against many pathogenic bacteria. LAB also have the ability to adhere tightly to the mucosa probably due to antigenic structures which recognize analogous structure on the mucosa and they spread out and settle in tracts of the intestine where they find the most suitable condition for development.

They are generally found in larger quantities in conditions where the content of the lumen moves at a slower rate than the rate of multiplication of the bacterial strain. The level of acidity in the stomach and the presence of oxygen favour the facultative anaerobes such as Lactobacilli and streptococci although, generally speaking, in this very hard environment growth is very limited. Facultative anaerobics prevail in the duodenum although they are hindered by bile salt and intensive peristalsis.

The presence of strictly anaerobic species such as Eubacterium and Bifidobacterium are more apparent in the distal part of the small intestine and in the caecum, due to the lack of oxygen.


A complex interaction exists between LAB, intestine and substances that are introduced in the intestine (e.g. food, antibiotics). The microflora works on the food bolus transforming it through its enzymatic system; however it may only be efficient if the number of bacteria is high (around 106/g). Due to the complexity of this interaction the diet and the intake of any other products may affect LAB equilibrium. The use of antibiotics for instance, modifies the ecological system by favoring the proliferation of resistant microorganism species, which become predominant and responsible for intestinal infections.


It is evident that modifications of the microflora may affect more than one bacterial strain, and therefore diseases in the upper part of the gut can be reflected in the lower part and vice versa. This implies that both therapeutical and prophylactic treatment should be considered in the use of different types of LAB and to aim to cover as many species as possible in order to restore microflora in the intestinal tract.

Based on these ideas, microbial intestinal disorders have traditionally been treated via the use of cultured diary products and more recently with viable LAB in order to supply the widest range of probiotics possible.


Production of bacteriocins
Bacteriocins are proteins or protein-particulate complex with bactericidal activities directed against species, which are closely related to the producer bacterium. The inhibitory activity of Lactobacilli towards putrefactive organisms is thought to be partially due to the production of bacteriocins. Some of the bacteriocins isolated from Lactobacilli are listed in Table 4.2


Production of other antagonistic substances
Lactic acid bacteria also inhibit the growth of harmful putrefactive microorganisms through other metabolic products such as hydrogen peroxide, carbon dioxide and diacetyl.

Table 2.2: Bacteriocins isolated from Lactobacilli

B-vitamins synthesis
Friend et al., reported that the B-vitamin content of fermented milk products was a function of species as well as the strain of lactic acid bacteria used in their manufacture. Although vitamins are synthesized by the lactic acid bacteria cultures in the gut microflora, in symbiosis with other flora, experiments on fermented milk products have revealed that lactic acid bacteria cultures require B-vitamins for their metabolic activities.


It has been observed that the diet of the host influences the nature and levels of beneficial intestinal microflora, such as Lactobacilli. The presence of dietary fructo oligosaccharides was found to enhance the nutritional effects of intestinal lactic acid bacteria. These compounds, found naturally in foods such as onion, edible burdock and wheat, are effectively employed as non-nutritive sweeteners (Neosugar, Meiologo). They have the advantage of being non-digestible by humans and farm animals, rendering them valuable in dietetic products. Intestinal lactic acid bacteria, especially bifido bacteria, thereby enhancing the beneficial effects of the intestinal flora, however, selectively utilize them.


Nutritional Benefits
Studies on rats have shown improved growth rate and increased feed efficiency when the rats were fed with yogurt containing Lactobacilli. Improved feed efficiency in rabbits fed with L.sporogenes supplemented diets have been reported. Although several Lactobacilli require B-vitamins for growth, some of these organisms are capable of synthesizing B-vitamins. The levels of some of the B-vitamins in yogurt are shown in the Figures 2.3. & 2.3a. Similarly, bioavailability of copper, iron, calcium, zinc, manganese and phosphorus was increased in yogurt-fed rats, suggesting that Lactobacilli facilitate nutrient uptake.

Therapeutic Benefits
Earlier research on indigenous microflora in animals and humans has shown their host-specificity and location-specificity, complexity in composition and their beneficial effects on the hosts.

Preparations containing Lactobacilli have been shown to be effective in the treatment of a variety of disorders and infections including colitis, constipation, diarrhea, recolonization of the intestine with pathogens after broad-spectrum antibiotic therapy, flatulence, acidity, hepatic encephalopathy, tumorigenesis, hypercholesterolemia, headache and vaginitis.

Fig 2.4 Hypocholesteromic activity of Lactobacilli

Probable mode of action

• Lactobacilli exert antibacterial activity against E.coli. Salmonella, Shigella, proteus, Staphylococci, Clostridia, etc and yeasts such as Candida sp., through the production of Lactic acid.
• Lactic acid produced by the bacilli helps enhancing the utilization of essential minerals such as Calcium, Phosphorus and Iron.
• Lactic acid produced stimulates the bowel movement and therefore relieves constipation.
• Lactobacilli produce mainly the vitamin B complex.
• Lactobacilli produce protein phosphoprotein phosphate, which digests the human milk protein, which is a property useful in infant nutrition.
• Lactobacilli control production of ammonia toxic amines, phenols and hydrogen sulfide.
• Lactobacilli control the diarrhea due to antibiotics.

• Cells are long and slender (0.3 to 0.8 pH), some are bent and all the cells have rounded ends.
• Motile with peritrichous flagellae
• Grain positive
• Colonies are usually 2.5 min in diameter, convex, smooth, glistening and do not produce any pigment.
• Extremely fastidious organisms requiring complex organic substrates for growth such as fermentable carbohydrate, peptone, meat and yeast extract. MRS medium supplemented with tomato juice, manganese, acetate and Tween-80 is a suitable medium for growth.
• Grow optimally at 300 C to 37 C and the optimum pH in the range 5.5 to 6.2
• Microaerophilic, exhibit fermentative metabolism and are facultatively aerobic
• Produce acid from arabinose, xylose, glucose, galactose, mannose, fructose, maltose, sucrose and trehalose
• Do not hydrolyse starch or casein
• Do not liquefy gelatin
• Are indole negative and do not produce hydrogen sulfide or gas.
• Produce L (+). (Dextrorotatory) lactic acid from glucose, fructose, sucrose, trehalose and inulin. Menaquinones are absent.

The characteristics of L. sporogenes as cited in Bergey's Manual (Seventh Edition) and other sources are: "Gram-positive spore-forming rods 0.9 by 3.0 to 5.0 micron size, aerobic to microaerophilic, producing L (+)-(dextrorotatory) lactic acid homofermentatively." Since L. sporogenes exhibits characteristics typical of both genera Lactobacillus, and Bacillus, its taxonomic position between the families Lactobacillaceae and Bacillaceae has often been discussed. This, along with the fact that there is no universally accepted official classification leaves room for controversy in the nomenclature". Some authors refer to L. sporogenes as Bacillus coagulans, although there seems to be no documented similarity between these organisms". The differentiation characteristics of L. sporogenes are indicated in Table 5.1

Table 3.1 L. sporogenes - Key characteristics for differentiation

However, L. sporogenes shares several characteristics with lactic acid producing organisms from the genus Lactobacillus. Therefore L. sporogenes is assigned to, the genus Lactobacillus.

L. sporogenes grows in the temperature range of 350C to 500C; the optimum pH range is 5.5-6.5. Unlike other Lactobacilli currently in clinical use, L. sporogenes, can form spores. Sporulation is the development in microorganisms of bodies each wrapped in a protective coat (a natural process of microencapsulation in 1 a calcium-dipicolinic acid- peptidoglycan complex). Under favorable, conditions, the spores germinate into viable bacilli and carry on their life activities. The spores of L.sporogenes are ellipsoidal bodies measuring 0.9 to 1.2 ' by 1.0 to 1.7 microns. Their morphology and formation are schematically, represented in Figures 3.1 & 3.2

Figure 3.1 Schematic representation of a bacterial spore

Figure 3.2 Diagrammatic Representation; Cytological changes that take place during the formation of the bacterial endospore

This property of spore formation by L. sporogenes is the main characteristic that makes it the probiotic of choice in clinical applications. It can survive changing microecological conditions better. Spores can tide unfavorable conditions and germinate when favorable conditions arise. On oral administration, these spores survive the acidic gastric environment and are activated due to the low pH, mechanical churning action of the stomach and the water in the gastric environment. The spore coats imbibe water, swell, and the increased water content causes a rise in the metabolic rate of the sporulated bacilli. Outgrowths begin to protrude from the spore-coats. The spores pass on to the duodenum where the outgrown cells germinate and transform into viable vegetative cells. They begin to proliferate in the small intestine, multiplying rapidly. Usually, germination takes place about four hours after ingestion. A large supply of viable L. sporogenes is thereby ensured in the small intestine, These cells settle in the intestinal tract and continue their metabolic activities, producing lactic acid and probably bacteriocins which render the intestinal environment non-conducive for the growth of harmful pathogenic bacteria". The maintenance of a low, constant level of lactic acid on the inner surface of the intestinal tract helps restore the microecological balance after antibiotic therapy. Antibiotic therapy may kill beneficial microbes, which help in the synthesis of B-vitamins and digestive enzymes. Since L. sporogenes produces only L (+)-lactic acid"', it does not cause metabolic acidosis.

Morphological and physiological characteristics of L sporogenes: The vegetative cells are rods occurring singly, rarely in short chains, the filaments Varying with cultural conditions. The cells are motiIe by means of flagella.

Table 3.2Carbohydrate fermentatio

Importance of viable bacterial count

The number of bacteria present in each capsule preparation is between 1.0-1.5 x 109 for each type, and is empirically based on the daily intake of yogurt or similar dairy products.

The standard concentration of single bacteria in yogurt should be >106/g although by the time they are ingested the concentration of bacteria is much lower. Bacteria do not reproduce and only a few survive once outside an ideal ecosystem. Survival of bacteria is much higher in dry media than in yogurt and is related to temperature and humidity factors. The latter should particularly be avoided with suitable manufacturing techniques.

A rational explanation for faulty counts on L. acidophilus is as follows:

L. acidophilus cells may not survive lyophilization. The freeze-dried cultures have to be stored under refrigeration and do not retain viability under normal conditions. Therefore, with time and fluctuations in storage conditions, there may be a fall in viable cell count.

LAB concentrations were tested in patients with different gastrointestinal diseases and were seen to be effective in the treatment of diarrhea and enteritis.


Importance of the bacterial mixture
Intestinal diseases, which are caused by microflora modifications and thus lead to infections, are not localized only in a specific area of the intestine, but usually spread out to both upper an lower part of the intestine. Hence, treatment should aim to restore general levels of microflora, and should be completed by the use of probiotics, which colonize various part of the gut.

The survival of probiotics depends on the production of metabolic end products by other genera, but may require continuous supplies until the moment they start colonizing and producing substantial amounts of acid compounds and bacteriocins.

LAB can be kept alive by addition of L. sporogenes as they feed on the peptoglycans of the spores. These two reasons highlight the importance of administering a combination of different probiotics in food supplement preparations.


Lactobacillus: Unique "Native" Bacterium in Human GI Tract
Lactobacilli constitute a major part of the microflora throughout the gastrointestinal tract. These bacteria have been proposed as candidate probiotic microorganisms to reinforce the barrier effect in the gut. Consumption of probiotic bacteria can alleviate intestinal inflammation, normalize increased intestinal permeability, and strengthen the intestine’s immunologic barrier function.

Lactobacillus (Lactobacillus, strain ATCC 53103) is a bacterium that occurs naturally in the human digestive tract. This strain of bacteria was first isolated by two Boston scientists, Professors Sherwood Gorbach and Barry Goldin, who were searching for a strain of Lactobacillus that could colonize the human intestine and thereby exert the beneficial effects which Metchnikoff had hoped to produce by his yogurt cultures. As guides for their research, Gorbach and Goldin established a number of criteria, which they believed their ideal probiotic candidate should satisfy.


Lactobacillus sporogenes -the ultimate probiotic
Lactobacillus sporogenes inhibits the growth of various pathogenic (disease causing) organisms including Candida, Fungal infections, Escherichia coli & others. Help lower serum cholesterol. Probiotics will help eliminate toxins, mucous & boost the immune system.

• The ideal bacterium would be:
• Be of human origin;
• Non pathogenic to humans
• Capable of attaching to human intestinal (epithelial) cells and colonizing the gut to prevent Competition from invading pathogens;
• Resistant to acid and bile, able to survive transit from the stomach to the intestines;
• Producing L (+) lactic acid during fermentation.
• High survival rate through processing conditions (during harvesting, drying etc.)
• High stability at room temperature separately or when mixed with other ingredients.
• Lack of potential to mate with potentially pathogenic microbes.
• Exhibit beneficial, health-promoting activity in the host system; and
• Exhibit a high degree of safety.