Probiotic Bacteria: Detection and Estimation in Fermented and Nonfermented Dairy Products W Kneifel and KJ Domig, BOKU – University of Natural Resources and Life Sciences, Vienna, Austria Ó 2014 Elsevier Ltd. All rights reserved. This article is a revision of the previous edition article by Wolfgang Kneifel, Tina Mattila-Sandhom, Atte von Wright, volume 3, pp. 1783–1789, Ó 1999, Elsevier Ltd.
Introduction According to deﬁnition, probiotics are live microorganisms, which when administered in adequate amounts exert beneﬁcial effects to humans and animals (see Microﬂora of the Intestine: Biology of Biﬁdobacteria, Biology of Lactobacillus Acidophilus, Microﬂora of the Intestine: Biology of the Enterococcus Spp., Microﬂora of the Intestine: Detection and Enumeration of Probiotic Cultures). Hence, the beneﬁcial nature of microorganisms as well as their individual viability and growth performance can be regarded as crucial factors in determining the microbiological and biofunctional quality of probiotic products. Probiotic microorganisms are the result of extensive selection and screening procedures aiming not only at identifying and assessing bacteria with deﬁned probiotic properties, which may vary from strain to strain, but also at ﬁnding strains with proven identity and safety as well as stability. Importantly, probiotic products have to pass clinical studies if they are to be marketed along with certain health claims. Hence, four main criteria comprise the general requirements for probiotics used in dairy products and thus form the basis for individual quality assessment of each product (Figure 1). Among these prerequisites, the bacterial viable count reﬂects the microbial cell density of a product and also the individual stability of the probiotic microorganisms used. The number of probiotic microorganisms in a product is usually examined with culture methods and expressed as colony-forming units per milliliter or gram (cfu ml 1 or g 1). High viable count levels are of major relevance for product quality and have to be maintained both during the shelf life and during gastrointestinal passage, upon ingestion of the product. These criteria are in accordance with the World Health Organization’s guidelines and are also taken into consideration by many legal authorities worldwide.
Microflora of Probiotic Products Individual Composition Different types of probiotic foods have been successfully developed and introduced as carriers of beneﬁcial microorganisms. Among these, fermented milk products (mainly yogurts and yogurt-like drinks) possess a major relevance in human nutrition, and a steadily increasing variety of probiotic yogurts has been offered on the market (see Fermented Milks: Range of Products). In addition, powdered infant formulas with viable probiotics embedded in a dry matrix also play some role as nonfermented products. It should not be forgotten that probiotic microorganisms have a long history as beneﬁcial feed additives. While different genera lactic acid bacteria (see Lactobacillus: Introduction, Lactobacillus : Lactobacillus delbrueckii ssp. bulgaricus, Lactobacillus: Lactobacillus Acidophilus, Lactobacillus:
Lactobacillus Casei, Lactococcus: Introduction, Lactococcus: Lactococcus lactis Subspecies lactis and cremoris, Starter Cultures, Streptococcus: Introduction, Streptococcus thermophilus) and biﬁdobacteria (see Biﬁdobacterium) dominate in probiotic foods and pharmaceutical preparations, other microorganisms such as bacilli and yeast have been applied in the feed industry (Table 1). Importantly, deﬁned strains with proven clinical evidence are preferred and marketed. Usually, the microﬂora of a probiotic yogurt is of dual-type composition and consists of different cultures, each with different tasks (Figure 2). The fermentation culture (mainly of Viable count and bacterial stability
Biofunctionality (probiotic effect)
Bacterial strain safety
Quality criteria of probiotic dairy products.
Table 1 Spectrum of microorganisms (genus level) relevant for probiotic products Food Lactobacillus, Bifidobacterium, Lactococcus, Streptococcus Pharmaceutical products and food supplements Lactobacillus, Bifidobacterium, Lactococcus, Streptococcus, Enterococcus, Escherichia coli, Saccharomyces Feed additives Lactobacillus, Bifidobacterium, Enterococcus, Pediococcus, Bacillus, Kluyveromyces, Saccharomyces
Figure 2 General microbial composition of probiotic dairy products (example fermented milk).
Encyclopedia of Food Microbiology, Volume 3
Probiotic Bacteria: Detection and Estimation in Fermented and Nonfermented Dairy Products
classical yogurt type) basically carries out the acidiﬁcation of the milk, also accomplishing typical ﬂavor and textural properties of the end product. In the case of probiotics, it may practically be considered as the background microﬂora. The aim of the probiotic bacterial compound is to equip the product with health-promoting attributes. Some probiotic bacterial strains are applied together with the fermentation culture, thereby yielding some co-fermentation, whereas others, which do not tend to multiply during fermentation, may be added to the fermented product as bacterial concentrates (either in a deep-frozen stage or as lyophilisates) right after classical fermentation. As different targets are to be met, Table 2 summarizes the most relevant individual differences between classical fermentation and probiotic cultures.
Microbiological Examination Methods The detection and enumeration of probiotic bacteria in fermented dairy products is part of the regular quality assessment and, in general, can be subdivided into two categories: routine and advanced methods. Concomitantly, the laboratory equipment necessary to facilitate these analyses is different and may range from conventional microbiological tools, glassware, and disposable material to cutting-edge analytical technology.
Routine Methods Routine methods mainly include conventional culture-based plate count techniques using an array of selective media and incubation conditions tailored for the different target microorganisms (Table 3). Most of the methods and media have been
Major differences between fermentation and probiotic cultures
Origin Physiological properties
Isolates from fermented food – Low intestinal relevance – High fermentation capacity – Formation of aroma compounds – Preservation effect Low (except enzymes) Typical for fermented product Product oriented
Mainly isolates of human origin or fermented milk – High intestinal relevance – Lower fermentation capacity – Limited formation of aroma compounds – Limited preservation effect High (probiotic biofunctionality) Characteristic and sufﬁciently high to exert probiotic effects Product- and gut-oriented (gastrointestinal passage)
Nutritional relevance Viable count Viable count stability
Survey of culture methods applied for the presumptive detection and enumeration of probiotic bacteria in fermented dairy products
Probiotic microflora Lactobacillus acidophilus group (L. acidophilus, L. gasseri, L. johnsoni)
MRS agar supplemented with ciproﬂoxacin (10 mg l 1) and clindamycin (1 mg l 1), anaerobic incubation for 72 h at 37 C
ISO 20128: 2006 (IDF 192: 2006) Milk products – Enumeration of presumptive Lactobacillus acidophilus on a selective medium – Colony-count technique at 37 degrees C. Kneifel and Pacher (1993). International Dairy Journal 3, 277–291.
Lactobacillus casei group (L. casei, L. rhamnosus) Biﬁdobacteria (B. animalis subsp. lactis, B. longum, B. breve, B. infantis, B. adolescentis) Fermentation microflora Yogurt bacteria (Streptococcus thermophilus, Lactobacillus delbrueckii subsp. bulgaricus) Sour milk bacteria (Lactococcus lactis)
Rogosa agar supplemented with Xglua at 40 mg l 1, anaerobic incubation for 72 h at 37 C MRS agar supplemented with vancomycin (50 mg l 1), anaerobic incubation for 72 h at 37 C TOSb (10 g l 1)-propionate agar supplemented with MUPc at 50 mg l 1, anaerobic incubation for 72 h at 37 C
M17 agar (S. thermophilus), acidiﬁed MRS agar (L. delbr. subsp. bulgaricus) aerobic incubation (M17) for 48 h and anaerobic (MRS) for 72 h at 37 C M17 agar, aerobic incubation for 48 h at 30 C
XGlu: 5-Bromo-4-chloro-3-indolyl-b-D-glucopyranoside (chromogenic dye). TOS: trans-galactosylated oligosaccharide mixture (prebiotic carbohydrate compound). c MUP: Mupirocin lithium salt (antibiotic). b
Björneholm et al. (2003). Microbial Ecology in Health Disease 14 (Suppl. 3), 7–13. ISO 29981: 2010 (IDF 220: 2010) Milk products – Enumeration of presumptive biﬁdobacteria – Colony count technique at 37 degrees C. ISO 7889: 2003 (IDF 117: 2003) Yogurt – Enumeration of characteristic microorganisms – Colony-count technique at 37 degrees C. ISO 9232: 2003 (IDF 146: 2003) Yogurt – Identiﬁcation of characteristic microorganisms (Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus).
Probiotic Bacteria: Detection and Estimation in Fermented and Nonfermented Dairy Products
Molecular methods for the selective detection and identification of defined probiotic strains Culture-independent methods for the assessment of viable and non viable bacterial cultures
Culture methods for the selective detection and enumeration of probiotic bacteria Figure 3
Culture methods for the selective detection and enumeration of the typical fermentation microflora
Routine Quality Monitoring
Methodological approaches in quality monitoring of probiotic dairy products.
standardized by international organizations and are based on extensive collaborative trials, including expert laboratories and statistical evaluation of the results. Usually, typical colonies selectively detected on the agar plates need to be veriﬁed by useful procedures. So, in general, these plate count methods are to be considered as methods for the presumptive detection and enumeration of speciﬁed microorganisms. While microscopical and enzymatic methods have formerly been applied to individual colony isolates to conﬁrm the results (e.g., the fructose-6-phosphate-phosphoketolase assay for biﬁdobacteria and physiological tests using microarray proﬁles for lactobacilli),
Advanced Quality And Identity Assessment
molecular biological methods have been increasingly introduced as an alternative with high precision and selectivity. These methods will be described in more detail below. For routine analysis of probiotic microogranisms in nonfermented products, the possible need for bacterial resuscitation should be taken into consideration. This is, for example, of relevance when microencapsulated bacteria are to be examined. The nature of the diluent as well as the temperature and duration of the resuscitation procedure prior to performing microbiological examination may inﬂuence the viability status and the individual growth performance of the target strains.
Survey of advanced methods applied for the detection, enumeration, characterization, and typing of probiotic bacteria
Molecular enumeration methods Hybridization techniques PCR-based techniques Flow cytometric techniques
Fluorescence in-situ hybridization (FISH) Real-time quantitative PCR (RT qPCR) Different staining principles allow the live/dead differentiation and speciﬁc detection of cells Identiﬁcation based on phenotypic information Morphology-based techniques Cultivation on selective and elective media, microscopy Physiology-based techniques Different biochemical patterns Chemotaxonomic markers Cell wall compounds, total cellular proteins, enzyme patterns, etc. FTIR spectroscopy Identiﬁcation is based on reference data basis, data processing using special software (e.g., artiﬁcial neural networks) Mass spectrometry e.g., by matrix-assisted desorption ionization-time of ﬂight mass spectrometry (MALDI-TOF MS) Identiﬁcation based on genotypic information Probe-based techniques Hybridization of complementary synthetic oligonucleotides to bacterial DNA sequences (e.g., microarray analysis) Ribotyping Separation and identiﬁcation of rRNA genes (e.g., 16S rRNA) PCR-based methods Application of primers for genus-species- and strain-speciﬁc targets Sequencing Based on whole genome sequencing or deﬁned genes (e.g., 16S rRNA, rpoB, recA, tuf, hsp60), which are of taxonomic relevance Molecular typing methods PFGE Pulsed-ﬁeld gel electrophoresis (PFGE) of chromosomal DNA after restriction with rare-cutting enzymes PCR-based typing Random-ampliﬁed polymorphic DNA (RAPD), repetitive-sequence-based PCR (repPCR), ampliﬁed fragment length polymorphism (AFLP), ampliﬁed rRNA restriction analysis (ARDRA) MLST Multi-locus sequence typing (MLST): characterization of alleles present at different house-keeping gene loci SNP/INDEL Single-nucleotide polymorphism (SNP) and INDEL (insertion/deletion) a
Taxonomic resolutiona Species–strain Species–strain Genus–species–strain Taxonomic resolutiona Genus–species Genus–species Genus–species–strain Species–strain Genus–species Taxonomic resolutiona Genus–species Genus–species–strain Genus–species–strain Genus–species Taxonomic resolutiona Species–strain Genus–species–strain
The taxonomic resolution of the various techniques also depends on differences within deﬁned genera (e.g., to date, more than 170 species and 27 subspecies of Lactobacillus spp. have been deﬁned).
Probiotic Bacteria: Detection and Estimation in Fermented and Nonfermented Dairy Products
Microencapsulation or special coating treatments of lyophilized bacteria are known to maintain elevated cell counts in dried food matrices, such as infant formula, even over several months.
As visualized in Figure 3, the spectrum of methodologies applied depends on the practical needs and on individual questions to be answered. If a clear indication of the viability is needed (e.g., when a stability monitoring of probiotic bacteria in products or even during gastrointestinal passage is of interest), staining methods using ﬂuorogenic markers (live–dead stains) demonstrating the individual stage of bacterial cells have been introduced. Furthermore, ﬂuorescence in-situ hybridization (FISH) techniques allow some selective monitoring of deﬁned strains. Instrumentally, methods can be performed either manually (via microscopy) or (semi-) automatically using ﬂow cytometric equipment. In addition to the enumeration of probiotic microorganisms, their taxonomical identity should be proved by state-ofthe-art methods. In general, taxonomical investigations need to be based on pure bacterial isolates. This means that pure cultures have to be grown before they can be identiﬁed. Only a very limited number of techniques (e.g., FISH, species- and strain-speciﬁc PCR-based detection) are applicable for samples with mixed strains or even more complex matrices (e.g., fermented milk, intestinal, or fecal samples). Based on that, a series of phenotypic and genotypic techniques have become available for the identiﬁcation of probiotics, each of them exhibiting different levels of technical complexity and taxonomic resolution (Table 4). For a ﬁrst tentative classiﬁcation on the genus level, phenotypic methods are often favored. However, the usefulness of biochemical systems is fairly limited due to the high intraspeciﬁc phenotypic variability observed with some species. Many of the identiﬁcation databases linked to these biochemical systems, therefore, are not well documented and lack the relevant species information. Today’s modern taxonomy of microorganisms is mainly built on molecular data. These kinds of data are becoming increasingly available as a result of the improved recovery and the growing understanding of DNA and RNA. The ongoing progress in method development, including the analysis of small amounts of DNA and RNA as well as the handling of large and complex datasets (e.g., sequencing and typing data), has led to a deepened set of analytical methods with different taxonomic resolution. Therefore, the complete identiﬁcation of a microorganism requires a larger number of techniques facilitating the so-called polyphasic approach to bacterial systematics: The more data you have to compare, the more complete and accurate the identiﬁcation is. Recent developments display a trend toward molecular techniques based on DNA-sequence-based information (e.g., Multi-locus Sequence Typing, Single-Nucleotide Polymorphismbased typing), which is complemented by the development of sophisticated techniques based on phenotypic information
(e.g., Matrix-Assisted Desorption Ionization-Time Mass Spectrometry, FTIR-spectroscopy).
See also: Bifidobacterium; Biochemical and Modern Identiﬁcation Techniques: Microﬂoras of Fermented Foods; Fermented Milks: Range of Products; Fermented Milks and Yogurt; Northern European Fermented Milks; Fermented Milks/ Products of Eastern Europe and Asia; Lactobacillus: Introduction; Lactobacillus : Lactobacillus delbrueckii ssp. bulgaricus; Lactobacillus: Lactobacillus brevis; Lactobacillus: Lactobacillus acidophilus; Lactobacillus: Lactobacillus casei; Lactococcus: Introduction; Lactococcus: Lactococcus lactis Subspecies lactis and cremoris; Microﬂora of the Intestine: Biology of Biﬁdobacteria; Biology of Lactobacillus Acidophilus; Microﬂora of the Intestine: Biology of the Enterococcus Spp; Microﬂora of the Intestine: Detection and Enumeration of Probiotic Cultures; Starter Cultures; Starter Cultures: Importance of Selected Genera; Starter Cultures Employed in Cheesemaking; Streptococcus: Introduction; Streptococcus thermophilus; An Introduction to Molecular Biology (Omics) in Food Microbiology; Identiﬁcation Methods: Introduction; DNA Fingerprinting: Pulsed-Field Gel Electrophoresis for Subtyping of Foodborne Pathogens; Identiﬁcation Methods: DNA Fingerprinting: Restriction Fragment-Length Polymorphism; Bacteria RiboPrintÔ: A Realistic Strategy to Address Microbiological Issues outside of the Research Laboratory; Multilocus Sequence Typing of Food Microorganisms; Application of Single Nucleotide Polymorphisms–Based Typing for DNA Fingerprinting of Foodborne Bacteria; Identiﬁcation Methods and DNA Fingerprinting: Whole Genome Sequencing; Identiﬁcation Methods: Multilocus Enzyme Electrophoresis; Identiﬁcation Methods: Chromogenic Agars; Identiﬁcation Methods: Immunoassay; Identiﬁcation Methods: DNA Hybridization and DNA Microarrays for Detection and Identiﬁcation of Foodborne Bacterial Pathogens; Identiﬁcation Methods: Real-Time PCR; Identiﬁcation Methods: Culture-Independent Techniques; Viable but Nonculturable.
Further Reading Bischoff, S.C., 2009. Probiotica, Präbiotica und Synbiotica. Thieme Verlag, Stuttgart (in German). Felis, G.E., Dellaglio, F., 2007. Taxonomy of lactobacilli and biﬁdobacteria. Current Issues In Intestinal Microbiology 8, 44–61. Kneifel, W., Salminen, S., 2011. Probiotics and Health Claims. Wiley-Blackwell, Chichester. Vankerckhoven, V., Huys, G., Vancanneyt, M., Vael, C., Klare, I., Romond, M.B., Entenza, J.M., Moreillon, P., Wind, R.D., Knol, J., Wiertz, E., Pot, B., Vaughan, E.E., Kahlmeter, G., Goossens, H., 2008. Biosafety assessment of probiotics used for human consumption: recommendations from the EU-PROSAFE project. Trends In Food Science & Technology 19, 102–114. World Health Organization (FAO–WHO), 2006. Probiotics in Food: Health and Nutritional Properties and Guidelines of Evaluation FAO Nutrition Paper No. 85.