Probiotic microorganisms in food. Properties, benefits, safety and enumeration
Written by Michael Mullan
This section gives an overview of developments in this area including opportunities for novel functional foods. The rare involvement of lactobacilli and starter bacteria in human infections is mentioned and a summary of traditional microbiological approaches to the enumeration of probiotic bacteria is included.
The last major update to this article was published in February 2008, since then there have been a number of significant developments. These include the failure of major European dairy companies to obtain ratification by the EFSA of health claims for probiotic products, the deaths of patients on a probiotic trial in the Netherlands, evidence that perhaps some bacteria designated as probiotics may have the potential to aggravate allergies in neonates. Additionally one major researcher has questioned where any strain of Lb. acidophilus has been shown to meet the criteria for a probiotic! However, there has been other more positive research indicating that particular strains of bacteria, in particular lactic acid bacteria, do have the potential to enhance immunity, reduce allergy, and to alleviate distant site infection. This work has very clearly shown that dairy companies and others have a responsibility to use only well characterised strains that have been shown to have probiotic effects in medical trials. Interesting Reid (2007) has stated "a potential major problem for probiotics is the misuse of the term. This can arise from products being poorly manufactured, or being referred to as probiotic without any relevant documentation. The net effect, deleterious to the overall field of probiotics, might be that such products are found to be ineffective, when in fact they were not even probiotic in the first place." Interestingly there is now a growing consensus that there is a world-wide, critical shortage of well qualified food scientists and technologists in commercial food manufacturing. These developments will be taken into consideration in the next major update to this article.
The next major update will summarise recent work on the gut flora and how this complex flora is thought to influence health. Recent research suggests that the gut flora can influence mood e.g. depression and its modification may have the potential to influence body mass and obesity. The main evidence for the latter has come from animal studies and anecdotal accounts of the consequences of faecal microbiota transplants also known as a stool transplants. This work suggests very significant potential for new generation probiotic products.
Gut problems e.g. dyspepsia (indigestion) are common and the Internet has many sites advocating probiotics for treating a range of symptoms. This article, while it may be of interest to the general public, does not promote the medical use of commercial yoghurt products, none of which currently have EFSA endorsements as probiotics in Europe, to treat gastrointestinal problems. I am aware of people experiencing dyspepsia who treated their symptoms with commercial yoghurt products and subsequently found that they had a range of physical medical conditions ranging from ulcers, hiatus hernia to more malign conditions that required surgical intervention. I am positive about the potential health benefits of probiotics but urge readers with health issues to discuss their problems with physicians, who certainly in Europe, take care not to do harm before self treating with yoghurt type products.
What are probiotics?
Probiotics are live microbial, dietary supplements or food ingredients that have a beneficial effect on the host by influencing the composition and or metabolic activity of the flora of the gastrointestinal (GI) tract.
The concept of probiotics has evolved from the work of Metchnikoff (1908) although the term was probably first used by Lilley and Stillwell (1965)-see table 1. Metchnikoff postulated that the apparent longevity of Balkan peasants was due to their ingestion of milk fermented with Lb. delbrueckii subsp. bulgaricus.
He hypothesised that the growth of this lactobacillus in the GI- tract would displace other putrefactive bacteria, reduce the concentrations of toxins in the gut and thus improve health.
While ingestion of Lb. delbrueckii subsp. bulgaricus may result in heath benefits it is now known that this organism cannot survive in the GI- tract and therefore has limited potential for markedly altering the flora of the GI- tract. It is therefore not a probiotic. There is a significant body of research that indicates that ingestion of dairy products fermented with this organism are beneficial to health e.g. they may allow lactose intolerant individuals to tolerate the residual lactose in fermented milk.
Because of the inability of Lb. delbrueckii subsp. bulgaricus to survive in the GI- tract researchers have advocated other microorganisms as candidates for delivering a range of benefits to consumers.
Benefits claimed for the ingestion of probiotic bacteria
A range of benefits have been claimed including:
- Improved digestion of lactose and reduce intestinal bloating, flatulence and discomfort
- Prevention of traveller's diarrhoea
- Prevention of the potential outgrowth of spores of Clostridium botulinum in the GI-tract, the associated toxin production and a possible cause of sudden infant death syndrome (SID)
- Enhancing the immune system, improving resistance to infection and improving well-being
- Protection against certain types of cancer
- Lowering serum cholesterol levels and reducing the incidence of coronary heart disease
- Prevention or helping treat peptic ulcer disease
- Treating intractable diarrhoea following antibiotic therapy
- Reducing allergic inflammation
NOTE!! While these benefits are cited in the literature there is limited quantitative data for some of these claims. There are also studies indicating no or limited benefits for the consumption of products containing probiotic bacteria. Some of the clinical trials that were successful also used very high concentrations of probiotic bacteria.
The International Dairy Federation has recently published a bulletin summarising the evidence for the effect of starter and probiotic cultures on a range of diseases and disorders in humans. The bulletin No. 380/2003, contains a section written by Ouwehand et al. (2003) reviewing the evidence for clinical effects in an extensive range of conditions including lactose maldigestion, diarrhoea, immune modulation, inflammatory bowel disease, necrotising enterocolitis, irritable bowel syndrome, constipation, Helicobacter pylori infection, small bowel bacterial overgrowth, colorectal cancer, superficial bladder cancer, cervical cancer, breast cancer, allergy, serum cholesterol, blood pressure, coronary heart disease, urinary tract infection, upper respiratory tract and related infections.
The authors' concluded that that there was good evidence for relief of lactose maldigestion and a shortening of the duration of rotavirus infection. They also concluded that while there was good evidence for the immune modulation that the health benefits to the consumer still required further clarification. Ouwehand et al. (2003) also considered that there was now promising evidence for some other clinical effects e.g. anti-tumour activity in respect of superficial bladder cancer, management of food allergy in infants. They also considered that certain claimed effects; in particular the effects on serum cholesterol and on the relief of constipation were not sufficiently substantiated at present.
Ouwehand et al. (2003), in addition to others, have commented that there is a need to quantify the effects of feeding probiotics to healthy consumers.
Probiotic bacteria and other microorganisms
The main probiotic microorganisms used belong to the Bifidobacterium and Lactobacillus genera. Other bacteria and yeasts e.g. Saccharomyces boulardii have also been used. Please see the paper by Kalpana Dixit and D.N.Gandhi on on the 'Biotherapeutic properties of probiotic yeast Saccharomyces species in fermented dairy foods'. Bifidobacterium species and strains of Lb. acidophilus and Lb. casei are now used extensively. Enterococci are also used occasionally as probiotics.
Bifidobacterium species have received particular attention and their study provides many insights into the potential therapeutic applications of probiotic bacteria. These organisms predominate in the GI- tract of babies fed with human milk where they account for some 95% of the flora. Breast-fed infants have a much lower rate of GI- tract infections than babies fed on bovine or other milks. The contrast between the two groups of neonates in developing countries is particularly striking. It is believed that bifidobacteria are responsible for the resistance of breast-fed infants to enteric infections. The predominance of these bacteria is due to selective agents in meconium (the sterile fluid in the GI-tract of human neonates), human colostrum and human milk. These selective factors are known as 'bifidus growth factors'.
Kuhn and his colleagues isolated the bifidus factor in Germany in the early 1950s. The factor was found to consist of several large oligosaccharides. Interestingly Kuhn and his colleagues found milk from ruminants to be low in these complex carbohydrates. This introduces prebiotics.
While some enterococci have also been successfully used as human probiotics they are also recognised as important nosocomial,aquired infections which are a result of medical treatment in for example a hospital, pathogens causing bacteraemia, endocarditis and other infections. One of the best-studied enterococci used as a probiotic is E. faecium strain SF68. This strain is considered to be an alternative to antibiotics for the treatment of diarrhoea.
Prebiotics are non-digestible food ingredients that have the potential to benefit the host by selectively stimulating the growth of desired host organisms in the GI- tract. Thus the oligosaccharides present in human milk are prebiotics. Clinical trials have shown that several different oligosaccharides can be used to stimulate bifidobacteria in the GI- tract. These include inulin, fructo-oligosaccharides (FOS), galactooligosaccharide and lactulose. These studies have shown that oligosaccharides have the potential, when included in infant formula, to stimulate host bifidobacteria to grow to levels similar to those in the tracts of breast-fed babies.
Inulin and FOS are now the most widely used prebiotics. They belong to a widespread group of naturally occurring carbohydrates that occur widely in plants. Natural sources containing high concentrations of inulin include chicory, artichoke, onions, leeks and garlic.
Inulin is composed of a chain of some 20 fructose molecules attached to a terminal glucose molecule. Inulin can be hydrolysed enzymaticaly to give lower molecular weight chains of fructose molecules referred to as FOS.
Laboratory studies have generally shown that growth of most species of bifidobacteria, with the exception of B.infantis, is stimulated by inulin and FOS. Current information suggests that many pathogens grow less well on FOS and inulin than on glucose. It is possible to design branched oligofructose chains that simulate the growth of bifidobacteria and that are not utilised by particular pathogens. This is an area of research interest.
There is an increasing volume of literature on the dose-response effects of ingesting inulin and FOS. High intakes can result in bloating and flatulence in some subjects. Supplement intakes of less that 5 grams a day appear to be well tolerated. These were generally high enough to demonstrate significant bifidogenic effects in all subject groups. Some caution on FOS supplementation has been indicated by Guigoz et al.(2002) who found that FOS supplementation while stimulating the bifidobacteria flora in the elderly also appeared to stimulate growth of potentially harmful clostridia and Bacterïodes.
Because inulin is so widespread in nature it has has GRAS (generally regarded as safe) status; see also the U. S. Food and Drug Administration GRAS Notice No. GRN 000118. This means that as far as the majority of the population is concerned ingestion of physiologically normal concentrations of this natural carbohydrate will not result in harmful effects.
Nothing in life is totally safe and there are risks in consuming foods just as in walking across the road or in driving a car. Gay-Crosier et al.(2000) at the University Hospital in Geneva, reported that inulin had caused a serious allergic reaction(anaphylaxis) in a patient who had suffered allergic reactions from foods that contained inulin in four separate events within a two-year period. The reactions occurred three times after ingestion of inulin-containing foods. Although there were no other reports of inulin sensitivity at that time, the authors expressed concern about the widespread use of inulin in processed foods and appeared to suggest that inulin may be the cause of more food allergies than is currently recognised. This concern appers to have some justification. More recently Fux et. al. (2005) have reported an anaphylactic reaction after 4 minutes of inulin infusion during a carefully controlled experiment thus confirming that inulin has the potential to promote extreme allergic reactions. This paper is available as a free download from the Am J Physiol Renal Physiol. For general information on allergy related matters readers may find the FANN site to be of interest.
Interestingly Szilagyi (2004) has defined lactose as a conditional prebiotic and has argued "that in lactase nonpersistent subjects (lactose intolerant), lactose qualifies as a prebiotic." The paper by Nupur Goyal and D.N. Gandhi provides further information on the role of lactose and the growth of probiotic microorganisms.
Characteristics of bifidobacteria
Bifidobacteria are normally found in the GI- tract of humans and other animals. While there are a number of different species, and there are some difficulties in differentiating them, Bifidobacterium longum is widely used. Although bifidobacteria produce lactic acid they are not LAB. Bifidobacterium species belong to the Actinomyces subgroup of the gram-positive eubacteria. These bacteria generally stain gram-positive and have a rod like appearance. However, the rods tend to be 'clubbed'. 'Wild' strains in particular demonstrate sometimes very irregular rods with branching. Often 'y' shaped rods can be seen. Because of their microscopic appearance they are usually described as pleomorphic. While these bacteria do have a characteristic appearance, care needs to be taken in the use of this criterion when working with non-dairy bifidobacteria since Actinomyces spp. have a similar morphology and are resistant to some of the antibiotics used in selective media.
Unlike other probiotic bacteria, bifidobacteria are obligate anaerobes and care should be taken to exclude oxygen during culturing and enumeration. Their optimum growth temperature is around 37º to 41ºC. Bifidobacteria produce both lactic (L-isomer) and acetic acids from lactose.
Because of their carbohydrate metabolism, isolates belonging to the genus Bifidobacterium can be confirmed reasonably easily by the detection of fructose-6-phosphate phosphoketolase (F6PPK) in cellular extracts.
The characteristics of Lb. acidophilus and Lb. casei have been described previously.
Mechanisms postulated for the beneficial effects of probiotic bacteria
The best-documented therapeutic application of probiotics is in the prevention and treatment of diarrhoeal diseases. This includes rota virus-induced diarrhoea in infants and the elderly. There is also an increasing volume of literature, some of it citing highly significant positive effects in the alleviation of allergies including a range of skin conditions. This area will be kept under review and periodically updated.
A recent publication on the benefits of probiotics and perhaps the most comprehensive todate is a book entitled 'Functional dairy products' (2003). Edited by Tiina Mattila-Sandholm and Maria Saarela. Published by Woodhead Publishing Limited, Abington, Cambridge, England and CRC Press LLC, Bocca Raton Florida, USA.
The beneficial clinical effects have been ascribed to:
• Altered IG tract micro ecology
• Normalisation of previously abnormal intestinal permeability
• Enhancement of gut barrier properties
• Enhancement of intestinal immunoglobulin response (IgA).
Safety of probiotics
Starter LAB have a long record of safe use, as do bifidobacteria, and are generally regarded as safe.
Several Lactobacillus species, and also lactococci, have been identified as causal agents of infections in humans. However, these infections are rare and have tended to occur in patients with compromised immune systems e.g. individuals with HIV infections or on drug regimes to suppress the immune system.
Following the deaths of some 24 Dutch patients treated with probiotics who were suffering from pancreatitis the safety of probiotics has been questioned in the media. I will update this article as more information about what happened becomes available. In the meantime readers may wish to follow the discussions in the forum concerning the trial to determine the value of probiotics in treating pancreatitis.
There is an early review of the safety of these bacteria by Aguirre and Collins (1993) and a more recent study by Boyle, Robins-Browne and Tang (2006). While it would appear that some infections have arisen from patients' indigenous LAB, the possibility of infections arising from LAB consumed through fermented products can not be ignored.
LAB can produce two stereoisomers of lactic acid, D and L lactic acid. Since some LAB also have racemases, some strains will produce D/L lactate.
L-lactate is readily metabolised whereas the D-isomer is not. There have been concerns about infants, in particular, ingesting high levels of D-lactic acid and there is a maximum recommended intake level for D-lactate. Because Lb. acidophilus produces D-lactic acid there is some interest in using other probiotic lactobacilli or bifidobacteria in products for babies and young children.
Several cases of D-lactic acid acidosis have been described in patients that have had intestinal bypass surgery. This condition is associated with transient neurological symptoms including headaches, weakness and visual disturbances. The D-lactic acid acidosis has been shown to be due to the overgrowth of Lb. acidophilus in the small intestine, generally due to its selection by antibiotic therapy. As with babies and young children, people who have had jejunoileal bypass surgery should seek medical advice before taking probiotic products particularly those containing D-lactate producing bacteria.
Some LAB can produce biogenic amines such as putrescine, cadaverine, histamine, tyramine and 2-phenylethylamine. Some of these can cause unpleasant reactions (nausea, headaches, and respiratory disorders) including dangerously high blood pressure particularly in individuals with reduced monoamine oxidase (MAO) activity or those taking MAO inhibitors, an older class of antidepressant medication. Providing strains are screened properly biogenic amine formation should not be a problem.
Regardless of the safe record of using LAB and probiotics there is comparatively little data on their safe use in feeding babies and very young children. Hence it would seem sensible to exercise caution in using them with young children and if in any doubt to seek pediatric advice first. As stated earlier it might also be better to avoid using any probiotic that produces D-lactic acid e.g. Lb. acidophilus.
In the section on cheese starters mention was made of concerns about using enterococci as starters. One of the concerns is that enterococci resistant to vancomycin have been isolated. This resistance is due to their possession of a plasmid that contains vancomycin resistance genes. Vancomycin is an important antibiotic in that it one of the few treatments effective against a number of pathogens that are resistant to other antibiotics. Unfortunately the vancomycin resistance plasmid can be transferred to Staphylococcus aureus and probably other pathogens. This is another argument against using enterococci as starters.
Safety is an important area as far as probiotic use is concerned and one that will be kept under review. Readers requiring more information are recommended to download, 'Probiotic use in clinical practice: what are the risks?' by Boyle, Robins-Browne and Tang (2006). This article co-authored by pediatricians and immunologists and published in the American Journal of Clinical Nutrition provides a balanced, positive perspective on the use of probiotics. The documented cases of both fungal (yeasts) and bacterial sepsis arising from probiotic use are described. This paper should be helpful to anyone considering feeding neonates, babies, with probiotics.
Selection of probiotics
Not all strains of Lb. acidophilus, Bifidobacterium species and Lb. casei are suitable for use as probiotics. The following criteria have been used or suggested for use in strain selection: -
• Origin. Strain should have originated from the human GI-tract
• Safety. Strain should be non-pathogenic. It should also be sensitive to common antibiotics and not harbour antibiotic resistance or virulence plasmids. Additionally I would also wish to ensure that strains that produce biogenic amines are excluded.
• Withstand host's natural barriers. Essentially be able to survive transit through the GI tract. This will mean resistance to bile salts, low pH and proteases in initial in vitro screening.
• Adherence to intestinal epithelium. The ability to adhere to intestinal cells and effectively block sites that could be occupied by pathogens.
• Commercial propagation. Strain must be able to grow in under commercial conditions and should retain viability under normal commercial storage conditions.
• Functional properties. The strain should meet the definition of a probiotic in clinical trials!
Note the abilities of strains to withstand normal host defences and to adhere to intestinal epithelium cells, if genetically transferred to pathogens, would be additional matters for consideration in selecting safe probiotics.
Minimum concentration of probiotic required for beneficial effect
There is insufficient information to recommend a minimum concentration of probiotic microorganisms in fermented products for beneficial effect in humans. The necessary human studies have not been completed. It would be expected that strains will differ with regard to their ability to colonise and proliferate in the GI tract. The efficiency of any therapeutic effect would also be expected to be strain dependant. Hence it would be expected that in practice the minimum concentration of a probiotic microorganism required to demonstrate health promoting effects will be dependant on many factors. Presumably host factors, possibly not well understood at present, will also have an effect?
Nevertheless, figures have been quoted and daily or weekly quantities of probiotic products have been recommended.
The Fermented Milks and Lactic Acid Beverages Association in Japan have introduced a standard of a minimum of >1 x 107 CFU/ml or CFU/g viable probiotic cells for fresh dairy products (standard cited by Ishibashi and Shimamur, 1993). Shah (2000) amongst others has suggested a minimum viable number of 106 CFU/ml or gram but recommends 108 CFU/g to compensate for reduction through passage through the gut.
It is generally accepted, again without much relevant research, that at the point of consumption probiotic products should have a minimum concentration of >1 x 106 CFU/ml or gram and that a total of some 108 to 109 probiotic microorganisms should be consumed daily if therapeutic effects are to be realised.
Enumeration of probiotic bacteria
Consumers buy probiotic products on the understanding that the genus and species designations are correct and that high concentrations of viable bacteria are present.
Traditional dairy products including yoghurts and cheese are used to deliver probiotic bacteria. This means that enumeration protocols must ensure that the selective agar media inhibit the growth of lactococci, Str. thermophilus and Lb. delbrueckii subsp. bulgaricus. However, they must also allow the selective enumeration of probiotic bacteria. In addition these bacteria have demanding nutritional requirements with the consequence that complex and relatively undefined basal media must be used.
The selective agents used include antibiotics, carbohydrates, 'salts' and bile. These can be used to develop selective agar media that when used with the correct gaseous environment and incubation temperature can give reliable quality assurance information.
Gentamicin, nalidixic acid, and neomycin are amongst the antibiotics that can be used to inhibit the growth of particular probiotic bacteria or LAB. In general Bifidobacterium species are relatively resistant to gentamicin, nalidixic acid and sensitive to neomycin (although less sensitive than starter bacteria and Lb. casei) while Str. thermophilus is sensitive to nalidixic acid and neomycin. Lb. acidophilus and Lb. bulgaricus are sensitive to all three antibiotics. In general lactococci are sensitive to low concentrations of these agents. Lb. casei, Lb. acidophilus and Lb. bulgaricus, lactococci and Str. thermophilus are sensitive to mupirocin while bifidobacteria are resistant.
Media containing maltose, raffinose, salicin or melibiose and other carbohydrates have been studied for their potential to select for bifidobacteria or Lb. acidophilus. Media containing maltose will permit the growth of many probiotic bacteria but will not enable the growth of most strains of Str. thermophilus and Lb. delbrueckii subsp. bulgaricus.
Relatively low concentrations of sodium chloride and other salts can be used to prevent the growth of Str. thermophilus, Lb. delbrueckii subsp. bulgaricus and Lc. lactis subsp. cremoris. Lithium chloride has also been found to be a useful selective agent.
Lactococci, Str. thermophilus and Lb. delbrueckii subsp. bulgaricus are inhibited by bile salts.
Since most strains of Lb. casei, unlike other probiotics, will grow at 15ºC temperature of incubation has generally been used as a selective factor to differentiate this organism from other probiotic bacteria. During studies of NSLAB growth in cheese, the author and colleagues noted the very high salt (sodium chloride) tolerance of Lb. casei and developed modified media that facilitated the isolation of this bacterium.
Media for the isolation of probiotic bacteria.
Rogosa agar is very useful in studies of starter and probiotic bacteria. Bifidobacteria, Str. thermophilus, Lb. delbrueckii subsp. bulgaricus, and lactococci do not grow on this medium. Most strains of Lb. acidophilus generally grow on this medium and it is a good growth medium for Lb. casei. In fact it can be made into a fairly good selective medium for Lb. casei by the addition of salts.
Enterococci. There is a range of media available for the enumeration of enterococci. Some media are fairly selective and in addition include differential effects e.g. colonies of enterococci may exhibit a particular reaction compared with other bacteria present.
Growth on bile-esculin medium, followed by simple confirmatory tests including Gram staining, demonstration of Gram positive cocci in pairs and chains, catalase-negative or pseudocatalase positive, growth in 6.5% NaCl and at pH 9.6 is indicative of an enterococcus isolate. Note most enterococci can cleave the glycoside esculin to esculatin and glucose in the presence of bile; the use of 40% bile effectively reduces the number of false positives. Readers may find the forum posts on this group to be of interest.
Bifidobacteria. Teraguchi et al. (1978) first advocated the use of the selective agents neomycin, nalidixic acid, lithium chloride and paromomycine sulphate (NNLP) to enumerate bifidobacteria. L-cysteine was also required to give reducing conditions. Since this paper was published in Japanese, in the Japanese Journal of Bacteriology, Laroia and Martin (1991) published a translation in the Cultured Dairy Products Journal. Since then media containing NNLP e.g. MRS + NNLP have been used widely; L-cysteine is also required. The selective agents are filter-sterilised before addition to sterile basal agar. While an experienced laboratory worker should have no difficulty in preparing agar media containing NNLP, it seems obvious to state that it is essential to ensure that all the components are in solution prior to filter-sterilisation. I suspect that some of the problems that may occasionally occur with this medium are due to incorrect preparation of the NNLP additives. Special care is also required with some of the selective agents, for health and safety reasons e.g. Control of Substances Hazardous to Health - COSHH regulations. The medium works reasonably well but does have some limitations. Some bifidobacteria will not grow on this medium, probably because of sensitivity to neomycin. Since strains in probiotic products generally grow well on MRS+NNLP this is generally not a practical problem for product quality controllers. Also, some strains of Lb. acidophilus may grow on this medium. Providing the necessary controls are provided, this is not a major problem. Lb. acidophilus will grow both aerobically and anaerobically whereas bifidobacteria will only grow anaerobically. Subtraction methods can easily be developed to quantify the populations of bifidobacteria and Lb. acidophilus present if required.
More recently the use of media containing the antibiotic mupirocin has been reported. Wilkins-Chalgren agar containing mupirocin was developed by Rada and Koc (2000) for the enumeration of bifidobacteria in dairy products. This is based on the finding by Rada (1997) that these bacteria are relatively resistant to mupirocin. Increasingly MRS, supplemented with the reducing agent L-cysteine, is being used as the basal agar medium. Care needs to be taken when using mupirocin when the environment contains Actinomyces spp; they have a similar morphology to bifidobacteria and are also resistant to muprirocin. Lactobacillus mucosae has also been reported to be resistant.
Mupirocin, in quantities necessary for bulk media production, is not easy to obtain. There are also ethical issues concerning the use of this antibiotic. It is particularly useful in the treatment of MRSA, methicillin-resistant Staphylococcus aureus. MRSA infection is a significant threat to the health of elderly and immunodeficient patients and has become a challenge to National Health Services in Britian and other countries. Mupirocin is also active against Helicobacter pylori, another bacterium that is difficult to treat using conventional antibiotic therapy.
Lb. acidophilus. MRS + Maltose agar (Hull and Roberts, 1984) is useful in the selective enumeration of Lb. acidophilus. While bifidobacteria can grown on this medium, they require anaerobic conditions and the inclusion of cysteine. Generally many of the Lb. acidophilus strains in yoghurt will grow aerobically on this medium. This medium has a major limitation in that it also permits the growth of Lb. casei, if this is present. Again providing good experimental design is used this is not a problem. Many strains of Lb. casei will grow well on MRS + Maltose agar + 4% salt while Lb. acidophilus does not grow. Again count-substraction methods can be developed to quantify both species. We have also found that some strains of of Lb. delbrueckii subsp. bulgaricus will also grow on this medium.
Lb. casei. Relatively little work has been published on the development of selective agar media for this bacterium. In fact it frequently grows, if present, in selective agars for other probiotic bacteria. While most strains will not grow on media containing NNLP e.g. MRS+NNLP, some strains may grow. Growth at 15ºC has generally been used along with subtraction counts. The use of MRS+ 4% salt and other media containing 4% salt can be regarded as being selective for this species providing one is working with dairy probiotic products containing only Str. thermophilus, Lb. delbrueckii subsp. bulgaricus, Lb. acidophilus and bifidobacteria. Note at laest one commercial dairy strain is inhibited by about 50% in the presence of 2% (w/v) sodium chloride in growth media and is inhibited by about 100% in media containing 4% salt. This strain is supplied by a major European culture supplier and is used in many dairy products. This justifies the caution mentioned below in using traditional microbiological methods to isolate and enumerate bacteria or other microorganisms.
Readers may be interested in the paper by Tharmaraj and Shah (2003) on the selective enumeration of Lactobacillus delbrueckii ssp. bulgaricus, Streptococcus thermophilus, Lactobacillus acidophilus, bifidobacteria, Lactobacillus casei, Lactobacillus rhamnosus, and propionibacteria. The paper is published in the J. Dairy Science (86:2288-2296).
Caution! While selective and differential agar are very useful in the isolation and enumeration of probiotic bacteria, care should be taken in the interpretation of the results (see the author's comments above); some LAB and probiotic bacteria may exhibit atypical reactions on these media. Consequently, the determination of other differentiating criteria including Gram reaction, morphology, the isomer of lactic acid produced, carbohydrate fermentation profile and the presence of F6PPK activity in cell extracts may be helpful in confirming identify. Currently the use of gene probes combined with PCR techniques are being developed.
Survival of probiotic bacteria in commercial yoghurt products
The author has investigated the survival of Lb. casei, Lb. acidophilus and Bifidobacterium species in commercial yoghurts over several years.
Apart from strain selection, perhaps the single most important factor affecting survival is acidity of the product. In general the more acidic the product, and the more the pH drops during storage, the lower the survival rate will be for intestinal-bifidobacteria and Lb. acidophilus. This means that yoghurt starters containing probiotic bacteria need to be 'adjusted'. In practice this means either removing Lb. delbrueckii subsp. bulgaricus, using lower-acid-producing variants or significantly reducing its initial inoculum level.
In our studies we have found genus and species designation to be correct. However, in some instances we failed to detect the presence of bifidobacteria although they were advertised as present in the product. Generally we have found low levels of survival of both Lb. acidophilus and Bifidobacterium species in many commercial products. Some of this information will be made available in this section.
Some product development considerations
This is a particularly active area. While yoghurt and cheese have been used as delivery 'vehicles' for both probiotics and prebiotics other product areas e.g. ice-cream, spreads, meat products, chocolate, liquid milks are being actively investigated. Attention is also being given to maintaining the viability of probiotics using modified LAB and new technology including immobilising the bacteria in a protective matrix.
There is also considerable interest in developing products containing prebiotics and several manufacturers are adding inulin and FOS to products.
European Community Regulation no 1924/2006 and health and nutrition claims
A new European regulation, Regulation no 1924/2006 , has been issued to help protect the public from false or misleading nutritional and health claims. The regulation requires that nutrition and health claims be authorised and added to the European Community (EC) list of permitted claims. The Food Standards Agency (FSA) is responsible for compiling the UK national list and is currently inviting food businesses to submit eligible claims for inclusion. This has relevance to companies producing probiotics and making health claims if they wish to continue making these claims.
The FSA website states that they will close this list on 21 September 2007.
The EC register of approved claims should be available by January 2010.
The FSA has provided a template to enable businesses to make claims and at the 13 th January, 2008 nutritional claims have been submitted for calcium, folate and iron.
The regulation will also result in the establishment of nutrient profiles for foods. Once these are established, health claims where any nutrient does not meet the criteria set by the nutrient profile for that food will not be permitted. It will be interesting to see how this works out for traditional foods high in fat for example, many cheeses, which provide major nutrients such as calcium.
Providing there is good scientific evidence it would appear to be relatively easy to make claims and businesses should benefit from reviewing the claims made to date.
Claire Towler has provided advice to companies using probiotics BB-12® and LA-5® supplied from Chr. Hansen who are considering making a nutritional or health claim through the FSA.
I will provide summary information on probiotic claims as they become available.
Probiotic and prebiotic reference list
How to cite this article
Mullan, W.M.A. (2002) .
[On-line]. Available from: https://www.dairyscience.info/probiotics/50-probiotics.html . Accessed: 26 April, 2019.
Revised 2004, December 2007, last revision February 2008, minor update December, 2008, minor update April 2015.