Chapter 2
Literature Review
2.1 The History of Probiotics
The concept of probiotics has been around for nearly 100 years. Yet its impact on human nutrition is still an emerging concept and there are much more to be explored. The word "probiotics" is derived from the Greek 'pro bios', which means 'for life' (Gismondo et al., 1999). In 1908, Eli Metchnikoff, Russian scientist found that the Bulgarian peasant gain remarkable longevity from the large consumption of fermented milk (Fioramonti et al., 2003). As such, Eli Metchnikoff carries out in depth investigation on this concept. His research began with the modification of the colonic microflora through the consumption of fermented milk (Fooks et al., 1999). Later, in the 1930s, Minoru Shirota claimed that good bacteria in the gastrointestinal tract might prevent disease (Brown et al., 2004). Since then, the concept of microbial functions and host health has gained much attention from the researchers (Fooks et al., 1999); (Gismondo et al., 1999).
In 1970s, researchers began to define "probiotic" and it was associated with microbial substances or live microbial feed supplement that improves microbial growth and give beneficial effects to the host (Schrezenmeir et al., 2001). The concept and definition of probiotics was periodically being revised. Eventually, the joint effort of Food Agricultural Organization and World Health Organization published the guidelines for probiotics application and now probiotics is termed as the viable microorganisms that may give health effects to human when administered in adequate amounts (Brown et al., 2004).
- Human Gastrointestinal Microbiota
The presence of microbes in the gastrointestinal tract has long been known and probiotics is recognized to have influence on the manipulation of the gut microbiota to improve the human health. Probiotics should be of human origin and thus it could be one of member in the gut microbiota. Within the human gastrointestinal tract, the microbial ecosystem is extremely complex and it encompasses more bacteria than the cells in the entire human body (O'Sullivan et al., 2005). There are approximately 500 different species of indigenous bacteria in the human large intestine. These microbes perform several functions, which in turn, affect the human health positively or negatively (O'Sullivan et al., 2005; Guarner et al., 2003; Vernazza et al., 2006).
The composition of the microbial community varied along the gastrointestinal tract (figure 2.1). Table 2.1 shows the microbial counts of different regions in the gastrointestinal tract and the factors affecting the microbial distribution (Vernazza et al., 2006). The environment in the mouth is considered aerobic. Then, the levels of oxygen start diminishing from oropharyngeal areas and progressively reduced down the gastrointestinal tract until the intestine where it becomes very anaerobic. The spread of oxygen along the gastrointestinal tract favor the specific microflora in each sections of gastrointestinal tract (figure 2.1). For instance, bifidobacteria are predominant in the anaerobic large intestine whereas the small intestine favors the microaerophilic Lactobacillus (Holzapfel et al., 1998). In the upper tract, the luminal medium such as acid, bile, pancreatic secretion kill most of the ingested microorganisms, and the activity of muscle contraction towards the ileal end hinder the colonization of bacteria in the lumen. On the other hand, the large intestine has a complex and dynamic microbial ecosystem with high densities of microbes, which up to 1011 or 1012 cells/g of luminal contents (Guarner et al., 2003).
Anatomical regions
Mouth
Stomach
Duodenum
Ileum
Colon
pH
6-7
1–3
Acidic–Neutral
Neutral–alkaline
5.5–7.2
Stasis
Periodic
Periodic
Propulsions
Prolonged
Prolonged/ retentive
Microenvironment
Teeth/ tongue
Mucus
Mucus
Mucus
Mucus/food/
epithelium
Approximate
number (cells/g
content)
108
10–100
10–1000
104–106
1012
Table 2.1: Population of microbiota in various regions of the human gastrointestinal tract (Vernazza et al., 2006).
Figure 2.1: Microbial colonization of the human gastrointestinal tract.
(Holzapfel et al., 1998)
Furthermore, it is reported that gut microflora are capable of performing several functions, which include metabolism of substances in gut and the protective function. In the gut, amino acids (arginine, cysteine and glutamine) and short chain fatty acids (acetate, propionate and butyrate) are the beneficial end products of the microbial fermentation of non-digestible carbohydrate residue. The short chain fatty acids is important to human as the energy source and influence the mucosal growth as well as blood flow in the colon. These effects will help in the inflammatory conditions of gut including ulcerative colitis, diversion colitis and pouchitis. Besides, gut microflora also involved in the synthesis of vitamins K and B complex, secondary bile acid production, neutralisation of dietary carcinogens such as nitrosamines. However, gut microflora also perform proteolysis of peptides and proteins that will produce toxic products such as ammonia, phenols, indoles and amines. Thus, diet has great influence on the gut health (O'Sullivan et al., 2005; Guarner et al., 2003).
Apart from the above effects, the indigenous intestinal microbiota act as a barrier against the transient potential pathogens by competing for nutrients, mucosal adherence and by local production of agents that is active against pathogens (O'Sullivan et al., 2005; Guarner et al., 2003).
Concurrently, a variety of potentially harmful microorganisms are members of the normal gut microflora. Under extreme condition, the gut microbiota will shift from the balance and away from potentially beneficial microbes such as lactobacilli and bifidobacteria, towards the potentially harmful microorganisms that may predispose to clinical disorders. Therefore, probiotics is claimed to be able to replenish the gut microbiota and improve microbial balance which in turn lower the risk of certain disease. A number of different factors are able to affect the composition of the colonic microbiota. Table 2.2 shows the factors that affect the microbial composition in gut (Fooks et al., 1999). Consequently, these harmful microorganisms or pathogens may be autochthonous within the intestinal niche, held in check by normal intestinal microbial activities and become pathogenic only when the gut ecosystem undergoes abnormal changes (O'Sullivan et al., 2005).
Table 2.2: Example of factors which may affect the composition of the human gut microflora.
Factors
1
2
3
4
5
6
7
8
9
10
Type of feeding or diet
Availability of microbial colonization sites
Immunological interactions
Intestinal transit time
Gut pH and redox potential
Availability of inorganic electron acceptors
Production of bacterial metabolites
Presence of antimicrobial compounds
Age of the host or human
Peristalsis
(Fooks et al., 1999).
- Colonization of Gut Flora
The stepwise microbial colonization of the intestine begins at birth and continues during the early phases of life to form an intestinal microbiota that is different for each individual subject. Establishment of the gut microbiota is characterized by the following steps: early colonization at birth with facultative anaerobes, such as the enterobacteria, coliforms, lactobacilli, and streptococci, first colonizing the intestine, followed by rapid succession of anaerobic genera, such as Bifidobacterium, Bacteroides, Clostridium, and Eubacterium (Salminen et al., 2006; O'Sullivan et al., 2005).
During and after the moment of childbirth, the fetus will expose to the numerous microbes from the maternal genital tract area and the surrounding environment. The microbes found in the surrounding environment may originate from air, nursing staff and neonatal care environments (O'Sullivan et al., 2005).
During the gut flora colonization period, some bacteria transiently colonize the gut whilst others survive and grow to form the indigenous microflora (Vernazza et al., 2006). Generally, intestinal bacteria can be divided on the basis of whether they can exert health promoting, benign or potentially harmful activities in their host (Vernazza et al., 2006). The pioneer bacteria will create a favorable habitat for themselves and can prevent growth of other bacteria introduced later in the ecosystem (Guarner et al., 2003). The initial colonization is therefore very relevant to the final composition of the permanent flora in adults (Guarner et al., 2003).
- Probiotics
Probiotics are found in human's gut and particularly in breastfed infants. Table 2.3 shows the example of probiotics. Most often, the probiotics come from the lactic acid bacteria, Lactobacillus and Bifidobacterium (Holzapfel et al., 2001).
Table 2.3: Species of Lactobacillus and
bifidobacteria used as probiotics.Lactobacillus species
Bifidobacterium species
Other lactic acid bacteria
L. acidophilus
B. adolescentis
Enterococcus faecalis
L. amylovorus
B. animalis
Enterococcus faecium
L. casei
B. bifidum
Lactococcus lactis
L. crispatus
B. breve
Leuconstoc mesenteroides
L. delbrueckii
B. infantis
Pediococcus acidilactici
L. rhamnosus
B. lactis
Sporolactobacillus inulinus
L. gallinarum
B. longum
Streptococcus thermophilus
L. gasseri
L. johnsonii
L. paracasei
L. plantarum
L. reuteri
(Holzapfel et al., 2001)
Lactic acid bacteria are Gram-positive bacteria and possess the characteristics as catalase-negative, microaerophilic to strictly anaerobic. The most important genera of lactic acid bacteria are Lactobacillus, Lactococcus, Enterococcus, Streptococcus, Pediococcus, Leuconostoc, Weissella, Carnobacterium, Tetragenococcus, and Bifidobacterium. Lactic acid bacteria are typically lactic acid producer when go through the carbohydrate fermentation (Klein et al., 1998).
2.4.1 Lactobacillus
Lactobacillus is characterized by the rod shape and the formation of lactic acid as the end product of carbohydrate metabolism. The Lactobacillus has complex nutritional requirements (carbohydrates, amino acids, peptides, fatty acid esters, salts, nucleic acid derivatives, vitamins) and may be either homofermentative (fermentative products as lactic acid) or heterofermentative (fermentative products include lactic acid, carbon dioxide, ethanol or acetic acid) (Tannock et al., 2004). The optimum growth occurs within 35-40°C but it can tolerate temperatures as high as 45°C. The optimum pH for growth is 5.5-6. Table 2.3 shows the species of Lactobacillus that used as probiotics which include L. acidophilus, L. casei, L. rhamnosus and L. plantarum.
In addition, L. acidophilus and L. casei are being the most widely use among the lactobacilli as they are heavily incorporated in probiotics products. Both of these species are acid tolerant and occur in high viability in commercial probiotic products (Liong et al., 2005). In this regard, it would valuable to selectively enumerate L. acidophilus and L. casei in assessing the quality of a product in term of viability as well as acid tolerant. Vinderola et al. (1999) formulated a differential medium for both the L. acidophilus and L. casei which are the MRS-bile agar. MRS-bile agar contains 0.15% of bile which enables the differential enumeration for both the L. acidophilus and L. casei by referring to the color of colonies. Likewise, Tharmaraj et al. (2003) provides the selective media for the enumeration of L. acidophilus and L. casei. Basal agar-sorbitol (BA-sorbitol) can inhibit other species of Lactobacillus while allow the growth of L. acidophilus at 37°C in anaerobic condition. On the other hand, MRS-vancomycine agar can inhibit other species of Lactobacillus while allow the growth of L. casei at 37°C in anaerobic condition (Tharmaraj et al., 2003). Consequently, MRS-bile is more practicable and feasible for the purpose of differential enumeration of L. acidophilus and L. casei since this media can reveal the colony counts of both of them (Vinderola et al., 1999; Vinderola et al., 2000).
2.4.2 Bifidobacteria
Bifidobacteria are Gram-positive, heterofermentative, non-motile and non-spore forming rods. Majority of bifidobacteria species are found in the gastrointestinal tract of mammals. Bifidobacteria produce acetate, lactate and small amounts of formic acid, ethanol as well as succinic acid during fermentation. Bifidobacteria display regular rod shape and various branched shapes (Y-shape). However, morphology of bifidobacteria cells may vary depending on species, culture conditions or growth phase (Klijn et al., 2005). The optimum pH for growth is 6-7 while optimum temperature for growth is 37-41°C.
In addition, bifidobacteria are emerging as probably one of the most important group of intestinal organisms with regard to human health. Yogurts with bifidobacteria have been launched for decades in Europe and Japan and their consumption is yet increasing (Roy, 2001). In this regard, differential enumeration of bifidobacteria is an important parameter in assessing the efficacy of this industrially important bifidobacteria.
Teraguchi et al. (1978) suggested that neomycin-paramomycin-nalidixic acid-lithium chloride with MRS agar as the basal medium (MRS-NNLP) is used as a selective media for enumeration of bifidobacteria. Bifidobacteria is shown to have good growth in MRS-NNLP agar with the exclusion of other lactic acid bacteria (Teraguchi, 1978). Neomycin sulphate and nalidixic acid act as the selective agent to inhibit the growth of Gram-positive and Gram-negative rods, respectively. Lithium chloride used for the selectively isolation of bifidobacteria in the presence of Lactobacillus. However, MRS-NNLP agar has certain limitation where it gives weak inhibitory effect on Streptococcus thermophilus (Roy, 2001). In this regard, MRS-NNLP agar is not suitable for assessing probiotic products that consist of S. thermophilus strains. On the other hand, Lapierre et al. (1992) developed another selective agar for bifidobacteria which contained lithium chloride and sodium propionate plus the liver-cystine-lactose (LCL) agar as the basal medium. This lithium propionate (LP) agar provides a simple and rapid way to enumerate bifidobacteria with the presence of lactobacillus since lesser ingredients are needed. However, LP-LCL agar has the same limitation as MRS-NNLP where it gives weak inhibitory effect on S. thermophilus (Lapierre et al., 1992).Consequently, MRS-LP was developed by Vinderola et al. (1999) as a differential medium to enumerate the bifidobacteria in the presence of Lactobacillus and S. thermophilus. MRS-LP contains 0.2% lithium chloride and 0.3% sodium propionate with MRS agar as basal medium. MRS-LP allows the growth of bifidobacteria
and L. casei with two different type colonies. Hence, MRS-LP allows the differential cell enumeration of bifidobacteria
and L. casei and this agar was selected for this study (Vinderola et al., 1999).
- Prophylactic and Therapeutic Uses of Probiotics
Recently, there are increasing numbers of experimental and clinical data to support the use of probiotics in prevention and treatment of various medical conditions. The uses of probiotics for its therapeutic effects include treatment of infectious diarrhea, antibiotic associated diarrhea, traveler's diarrhea, lactose intolerance and constipation by affecting the host cells and intestinal microbes (O'Sullivan et al, 2005; Brown et al., 2004).
2.5.1 Diarrhea
Diarrhea is a condition in which the sufferer has frequent bowel movements with watery and loose stools. Diarrhea causes accumulation and expulsion of fluid from the intestinal tracts which lead to loss of body fluid and electrolytes. Acute infectious diarrhea is a common cause of death (O'Sullivan et al, 2005); (Brown et al., 2004).
It was reported that the consumption of high level (1010 CFU/ day) of probiotics may shorten the duration of certain diarrhea illnesses. The sum of these therapeutic effects of probiotics is due to its ability to attract to enterocytes and colonize the crypts which leads to the competitive displacement of intestinal pathogens, cyctokine synthesis, interferons synthesis, cell resistance to viral attack and others (O'Sullivan et al., 2005).
Probiotics have been reported to treat three types of diarrhea which is antibiotic-associated, travelers, and infectious diarrhea (Brown et al., 2004). Antibiotic-associated diarrhea is defined as acute inflammation of the intestinal mucosa which is caused by the administration of broad-spectrum antibiotics (Kotowska et al., 2005). Antibiotics which are particularly active against anaerobes of gut flora will cause diarrhea. However, aminopenicillins, combination of aminopenicillins and clavulanate, cephalosporins, as well as clindamycin impose higher risk. The common side effect of antibiotic therapy is diarrhea (Szajewska et al., 2006).
According to Nomoto (2005), antibiotic may cause imbalance of intestinal microflora which lead to diarrhea. Thus, the rationale for the use of probiotics in antibiotic-associated diarrhea is based on the assumption that in some instances, diarrhea is associated with disturbance in the normal intestinal microflora (Surawicz, 2003).
The efficacy of probiotics in the treatment of antibiotic-associated diarrhea was investigated regularly by researchers. Brown et al. (2004) reviewed the studies on this field and concluded that
L. rhamnosus GG was effective for reducing the incidence of antibiotic-associated diarrhea.
In addition, two randomized controlled studies by Vanderhoof et al. (1999) and Arvola et al. (1999) also agreed to the usage of probiotics in the treatment of antibiotic-associated diarrhea. Both studies involved 307 participants and showed that the reduction of the risk antibiotic-associated diarrhea was associated with the use of L. rhamnosus GG (Vanderhoof et al., 1999); (Arvola et al., 1999).
Apart from L. rhamnosus GG, other Lactobacillus is also effective for the treatment of antibiotic-associated diarrhea. A meta-analysis investigated all studies in these related field between 1966 and 2000 and from all the outcome studies, nine studies which were randomized, double blind, placebo controlled trials of probiotics were evaluated. In all the nine studies, probiotics were given in combination with antibiotics and the control groups received placebo and antibiotics. It was concluded that S. boulardii and Lactobacillus were effective in the prevention of antibiotic-associated diarrhea (D'Souza et al., 2002).
Besides, a meta-analysis on children and infant was completed by Szajewska et al. (2006) which investigated different studies done up to the year 2005. Based on 9 studies, he concluded that there were evidence of effectiveness of probiotics in the prevention of antibiotics-associated diarrhea by S. boulardii, L. rhamnosus GG, B. lactis and S. thermophilus. The finding also showed that probiotics significantly reduced the risk of diarrhea in children treated with antibiotics in general (Szajewska et al., 2006).
The status of probiotics in the treatment and prevention of antibiotic associated diarrhea have a positive correlation (Saxelin et al., 2006). In addition, the results emerging from all the studies provide evidence of a moderate beneficial effect of probiotics in the prevention of antibiotic-associated diarrhea.
Traveler's diarrhea is one of the most common medical conditions experienced by individuals who travel to a developing country, underdeveloped or high-risk countries. Traveler's diarrhea may be acquired by ingestion of food, water or other liquids contaminated with fecal material. The period from the exposure to the contaminated food to the beginning of symptoms usually is 2 to 3 days (McFarland, 2005). The major symptom is diarrhea, where the traveler may experience up to 4 to 6 days of loose, watery or bloody bowel movements (McFarland, 2005). According to Brown et al. (2004), there were eight clinical trials ranging from 1978 to 1997 that investigated the effectiveness of orally administered probiotics to prevent traveler's diarrhea and the results showed that the occurrence of traveler's diarrhea decreased. Furthermore, it was reported that the mixture of L. acidophilus, L.s bulgaricus and B. bifidum reduced the incidence of traveler's diarrhea by restoring the intestinal homeostasis (Brown et al., 2004).
The use of probiotics may help travelers in minimizing their dependency on antibiotics. Moreover probiotics are generally well-tolerated, even for prolonged use. The main advantage of probiotic therapy for traveler's diarrhea is that they do not disrupt the re-establishment of the protective normal microbial flora. From the meta-analysis of probiotics for the prevention of traveler's diarrhea, twelve studies indicated that S. boulardii as well as mixture of L. acidophilus and B. bifidum significantly prevent the traveler's diarrhea (McFarland, 2005). In addition, a review by Nomoto (2005) suggested that L. rhamnosus GG
may also
decrease the incidence of traveler's diarrhea.
It was suggested that the selection of
appropriate traveling regions for trials would aid in obtaining more reliable results. Therefore, probiotics may offer a safe and effective method to prevent traveler's diarrhea.
In recent years, numerous studies had been performed on the role of probiotics' treatment of infectious diarrhea. For instance, Van Niel et al. (2002) suggested that Lactobacillus is an effective treatment for children with acute infectious diarrhea in his meta-analysis which took into account of all the randomized controlled trials from 1966 until 2000. In the analysis, nine of the studies met the inclusion criteria, which were blinded and controlled randomized trials. In all the included studies, the treatment group received Lactobacillus and the control group received placebo (Van Niel et al., 2002).
The common pathogens of infectious diarrhea are Shigella, Salmonella, Campylobacter, Colostridium difficile, rotavirus, enterotoxigenic Escherichia coli and Helicobacter pylori (Brown et al., 2004). Although the normal human microflora acts as the barrier against the colonization of exogenous pathogens, the probiotics are also known to contribute to this barrier function (Parassol et al., 2005). Essentially, probiotics were applied in such situation.
Helicobacter pylori
is a Gram-negative spiral-shaped, microaerophilic rod and causes gastritis and peptic ulcer. It is also suggested to be the risk factor of gastric cancer (Nomoto, 2005). Helicobacter pylori produced urease which will hydrolyse into ammonium and lead an increased pH in the stomach. These promote the colonization of the pathogenic microorganism (Sullivan et al., 2002).
It was reported that the administration of L. acidophilus, B. subtilis
and L. johnsonii La 1 may eradicate or inhibit the H. pylori
in infectious diarrhea (Sullivan et al., 2002; Cruchet et al., 2003). In addition, Sullivan et al., (2002) suggested that B. subtilis inhibit H. pylori by acting as a bactericide. Likewise, according to Cruchet et al. (2003), L. johnsonii La 1 would delay the colonization of H. pylori by restricting the size of the pathogen's population.
Other pathogens such as E. coli causes prolonged watery diarrhea in children. E. coli colonize the small bowel and adhere intimately to the apical enterocyte surface which will destroy the enterocyte microvilli. It also induced several changes in the host cell, such as increase in intracellular calcium concentration and phosphorylation of several proteins. Result of human studies showed that L. casei prevents acute infectious diarrhea (Parassol et al., 2005).
Rotavirus causes severe diarrhea in children and from double-blind, placebo controlled randomized studies, it showed that probiotic therapy especially Lactobacillus GG were effective in reducing the duration of rotavirus enteritis infection (O'Sullivan et al., 2005). The protective role of probiotics against intestinal colonization by pathogenic microorganisms has gained more credibility and may represent a new effective tool to control pathogen overgrowth inside the gastrointestinal tract of patients. The extent to which these in vitro results correspond to in vivo conditions remains to be determined; clinical trials are needed to assess the benefits of probiotic organism consumption in the management of these diseases.
2.5.2 Lactose Intolerance
Lactose intolerance present in people who have congenital deficiency of enzyme beta-galactosidase (also known as lactase) and lead to inability to digest lactose. The symptoms of lactose intolerance include diarrhea, bloating, abdominal pain and flatulence. In lactose intolerant people, undigested lactose in the large intestine is fermented by the colonic microbes that produce gases and products that lead to watery stool (Rolfe, 2000).
Moreover, lactic acid bacteria are showed to facilitate the digestion of lactose through fermentation and replication of the lactic acid bacteria itself in the gastrointestinal tract which will release lactase. Studies also showed that Lactobacillus casei NCFM is able to survive through the stomach passage and colonize the intestine and eventually reduce lactose intolerance (Rolfe, 2000).
In addition, lactose positive strains of bacteria such as Lactobacillus and Bifidobacterium were commonly added to pasteurize dairy products to increase the digestibility of lactose in the foods. L. acidphilus and Bifidobacterium produce beta-galactosidase which auto digests lactose and improve tolerance to lactose (Kailasapathy and Chin, 2000).
Apart from L. casei and Bifidobacterium, it is reported that L. bulgaricus and S. thermophilus also improve the digestion of lactose. The different strains and species of probiotic digest lactose more or less efficiently due to their varying activities of bacterial enzymes. Eventually, improved digestibility of lactose by probiotics was partially due to the activities of bacterial enzymes beta-galactosidase (Brown et al., 2004).
2.5.3 Constipation
Constipation refers to infrequent or hard stools, or difficulty passing stools. Constipation may involve pain during the passage of a bowel movement, inability to pass a bowel movement after straining or pushing for more than 10 minutes, or no bowel movements after more than 3 days. Infants who are still exclusively breastfed may go 7 days without a stool (Fernandez-Banares, 2006; O'Sullivan et al, 2005).
The primary causes of constipation are divided into three subtypes, including slow transit constipation, outlet delay constipation, and functional constipation. While, the secondary causes of chronic constipation are medications, endocrine or metabolic disorders, neurologic disorders and gastrointestinal disorders (Fernandez-Banares, 2006).
It was reported that L. acidophilus may reduce the occurrence of constipation by restoring the homeostasis of the gut microflora (O'Sullivan et al, 2005).
In colon, fiber or non-absorbed carbohydrates will be digested by colonic anaerobic bacterial flora which produces polysaccharidases and other enzymes. These are characterized by a complex series of inter-related reactions resulting in the formation of a variety of end-products including short-chain fatty acids (acetate, propionate, and butyrate), gases (hydrogen, methane, carbon dioxide), as well as energy, which bacteria use for growth and maintenance. It has been estimated that about 70 g carbohydrate per day is required to maintain the colonic flora (Fernandez-Banares, 2006).
In a double blind, randomized, controlled study, B. animalis is shown to be efficient in reducing the colonic transit time in a group of healthy women aged 18 to 45 years. Likewise, in an open trial of elderly subjects, a commercial mixture of L. rhamnosus and Propionibacterium freudenreichii were shown to improve the defecation frequency among the subjects (Ouwehand et al., 2002). Similarly, in a double blind, placebo-controlled, randomized study of 70 patients with chronic constipation showed that L. casei Shirota may relieve constipation (Koebnick et al., 2003). From the above studies, probiotics are found to be effective in patients with mild to moderate constipation.
2.6 Selection Criteria of Probiotics
2.6.1 Tolerance to Low pH
It has been reported that selection criteria of probiotics include capability to maintain their viability during processing and storage, tolerant to acid and bile, able to adhere to the intestinal epithelium of the hosts and possess antagonistic activity against bacterial pathogens. Besides, they should be generally recognized as safe (GRAS) (Lin et al., 2006). Ability to tolerate stomach acidity is among the criteria reported to be important. Before reaching the target site in the intestine, probiotics must first overcome the stress encounter in the stomach. In the stomach, the secretion of gastric acid constitutes a primary defense mechanism against most ingested microorganisms. As such, one of the first challenges encountered by probiotics following ingestion is the ability to tolerate in such acidic conditions to reach the distal part of intestine in sufficient numbers (>106 cells/g) in order to impart its health benefits. Furthermore, the gastric emptying rate of healthy individual should also be considered in such condition. Tolerance to acidity refers to a microorganism's ability to survive such acidic stress (Dunne et al., 2001; Ross et al., 2005; Tuomolo et al., 2001). Therefore, preliminary test was conducted to determine the acid tolerance exhibited by the pharmaceutical probiotics.
The stomach is physiologically acts as a reservoir that regulates the rate food entry and number of bacteria entering the small intestine. Human secrete approximately 2.5 to 3.0 liters of gastric juice each day, which contains hydrochloric acid as the first phase of gastric secretion and lead to the gastric pH of around 2. The presence of foods may increase the pH to 4.5 or slightly higher (pH 6). Besides, the amount of time food remains in the stomach depends on the food's composition and ranging from 90 minutes to several hours, with an average time of three hours (Waterman et al., 1998; Lin et al., 2006; Campbell, 1996). In this regard, this study will examine the acid tolerance of probiotics in the pH 2, 3, 4 and 6 within three hours.
Many studies have been conducted on the survival of probiotics in various pH levels in foods. Berrada et al. (1991) reported that bifidobacterium strains possess different degree of acid tolerance and response when exposed to an in vitro simulated gastric acidity. From his study, one strain survives very well but the second strain is much less resistant. These in vitro results, with slight differences, were validated by in vivo study in humans. The in vitro test was performed by adding acid hydrochloric (HCl) to the sample, fermented milk in order to obtain pH 3 and subsequently, after 5 and 90 minutes, inoculated in the trypticase phytone yeast (TPY) agar to obtain the total plate count. On the other hand, the in vivo test was performed by feeding 250g of the fermented milk to 10 healthy adults and the sample was taken via a gastric tube after 0, 30, 60 and 90 minutes after ingestion. Afterward, it was inoculated in TPY agar to obtain the total plate count. In addition, Berrada et al. (1991) also studied the rate of gastric emptying on 12 healthy individual, by randomly ingested 250 ml of fermented milk containing rhenium sulfur colloids, non absorbable by the digestive tract, labeled by radioactive isotopes. The result showed that stomach was half emptied within about 41 minutes to 41 minutes and over 80% of the stomach content was emptied after 90 minutes (Berrada et al., 1991).
Similarly, screening test of acid resistant in bifidobacteria
from human feces was completed by Chung et al. (1999). From the study, two selected Bifidobacterium spp. referred as HJ 30 and SI 31, exhibit higher rates of survival at pH 2 and pH 3 of HCl acidified phosphate buffer solution. Viability of SI 31 declined 100-fold and HJ 30 declined 1000-fold at pH 2 after 1 hour. Then, at pH 3.0, viability of SI 31 and HJ 30 exhibit 10-fold decline for 2 hours incubation (Chung et al., 1999). Another study by Vernazza et al. (2006) showed that bifidobacterium
spp. were poorly resistant to pH 2. The strains tested were Bifidobacterium adolescentis DSM20083, Bifidobacterium infantis DSM20088, Bifidobacterium longum DSM 20219, B. lactis Bb12, and B. longum 46. The study showed that for all of the strains tested except B. lactis Bb12 was lethal after only 10 minutes exposure at pH 2 (Vernazza et al., 2006).
Subsequently, Collado et al. (2006) reported that Bifidobacterium breve and
B. adolescentis were intrinsically resistant to acidic gastric conditions (pH 2.0). B. breve and
B. adolescentis were infant-type and adult-type species respectively, show highest ability to develop an acid-tolerant response. This suggests that infant-type bifidobacteria
are better adapted to stomach acidity whereas in aged individuals,
bifidobacteria
are not predominant but replaced by other anaerobes. Bifidobacterium
spp.
from adult samples might be older residents of the colon and therefore better adapted to its neutral environment than those from infant samples. However, the ability of the isolated bifidobacterial strains to survive the simulated gastric conditions also depends on the sample origin, species, and strains. In this regard, acid response of bifidobacteria
was varied widely, in accordance with Berrada et al. (1991) and Vernazza et al. (2006). Ultimately, the viability of bifidobacterial strains at pH values of gastric juices is generally considered to be low (Charteris et al., 1998; Matsumoto et al., 2004; Takahashi et al., 2004).
Furthermore, L. acidophilus was tested for tolerance to hydrochloric acid at pH 3.5 for 90 minutes and it showed that this strain have good acid tolerance. L. acidophilus was inoculated into MRS broth acidified with concentrated hydrochloric acid to pH 3.5 (Chou et al., 1999). Mishra et al. (2005) tested seven strains of L. casei for its acid tolerant with in vitro method. The cell suspension containing L. casei was added to pH 1.0, 2.0 and 3.0 in HCL adjustable double distilled water. The findings were none of the strains survived at pH 1 while only three strains (C1, Y and NCDC17) exhibited considerably good acid tolerance at pH 2 and 3. The survival of them was better at pH 3 and this characteristic is noteworthy as ingestion with food raises the pH in stomach to 3.0 or higher (Mishra et al., 2005).
In this study, three pharmaceutical probiotic products were selected for acid tolerant test simulating the pH of human stomach of by taking into account of the general stomach transit time. Besides, it is known that stomach pH varies according to individual and whether individual has fasted prior to ingestion. To account on these inter-individual differences, the ability of probiotic strains to survive at various pH was investigated. The test was designed to be consisted of four level of pH which were pH 2, 3, 4 and 6 with the stomach transit time of 0, 1.5 and 3.0 hours. Zero hour incubation of acid tolerance test was used as a control test to determine the changes in viability during the three hours of acid exposure.
2.6.2 Tolerance to Bile
The next criteria of probiotics would be the bile stability or tolerance to bile (Tuomola et al., 2001). This is the barrier that probiotics must overcome in the small intestine. The conditions of the small intestine include the presence of bile salts and pH of around pH 8. Moreover, the transit time of food through the small intestine is generally between 1 and 4 hours. Generally, tolerance to bile can be examined by testing their survivability in the presence of bile salt with concentration of 0.15–0.3% bile salt. This concentration has been used for selecting probiotics for human use (Huang et al., 2001).
Bile acids are synthesized from cholesterol in the liver and are secreted from the gall bladder into the duodenum in the conjugated form. Bile acids are then chemical modified by microbes in the colon. These activities included deconjugation, dehydroxylation, dehydrogenation, and deglucuronidation. Nevertheless, the deconjugated forms are inhibitory to certain Gram-positive bacteria and probiotics (Huang et al., 2001).
2.6.3 Adhesion to Intestinal Epithelial
For many of the criteria, adhesion of the probiotic to the intestinal mucosa is considered important for immune modulation, enhanced healing of damaged gastric mucosa, and antagonism against enteric pathogens. Adhesion of probiotic strains is variable. Adhesion of probiotics was studied by using a human colon carcinoma cell line (Caco-2) as in vitro models for intestinal epithelium. Adhesion of probiotic strains form the basis of human intestinal colonization which in turn its health effects (Tuomola et al., 2001).
No comments:
Post a Comment