Download Appendix XV: Microbial Food Cultures Including Probiotics

SAMPLE - FOR PROMOTIONAL USE ONLY
FCC 9
General Tests and Assays / Appendix XV / 1477
APPENDIX XV: MICROBIAL FOOD CULTURES INCLUDING PROBIOTICS
INTRODUCTION TO MICROBIAL FOOD
CULTURES
Holzapfel, W.H., U. Schillinger, and R. Geisen. “Biological Preservation of
Foods with Reference to Protective Cultures, Bacteriocins and Food-Grade Enzymes.” International Journal of Food Microbiology 24, no. 3 (January
1995):343–362.
1
Copyright United States Pharmacopeia, 2014. All rights reserved.
USES OF MICROBIAL FOOD CULTURES IN
FOOD PRODUCTION
MFC consist of one or more microbes and unavoidable media components carried over from the fermentation and
concentration process. In addition, ingredients may be
added to facilitate microbial survival during storage and to
standardize microbial cell concentration or activity. MFC are
used as food ingredients at one or more stages in the food
manufacturing process to attain specific sensorial, nutritive,
or stability properties of the fermented foods or, with regard
to probiotics, to confer purported health benefits.
MFC Used to Transform Foods MFC used to transform
foods are referred to as starter cultures. Starter cultures have
been defined as “preparations of living microorganisms in
their resting forms, whose metabolic activity has desired effects in the fermentation substrate food.”4 They are tools
used to transform historically uncontrolled fermentation
processes performed by indigenous microorganisms into
processes that are controlled with regard to predictability,
reproducibility, economy, timing, safety, and high standard
of food quality.
Starter cultures are commercially available for use in foods
such as dairy, meat, and wine fermentations and, to a lesser
extent as cultures for fish, vegetable, fruit, and dough fermentation. Furthermore, yeasts for production of beer, wine
spirits, and leavened bread are also appropriately regarded
as starter cultures. In Asia a multitude of cultures also exist,
such as for production of koji (used for soy sauce, sake or
vinegar) or red rice (also known as Ankak).
FAO. Health and nutritional properties of probiotics in food including
powder milk with live lactic acid bacteria. In Food and Agriculture Organization of the United Nations (FAO) [database online]. Cordoba, Argentina, 2001
[cited 5 March 2011]. Available from http://www.who.int/foodsafety/publications/fs_management/probiotics/en/.
3
Human Microbiome Project. Human microbiome project (HMP). 2011
[cited 5 March 2011]. Available from http://hmpdacc.org/.
4
Vogel, R. F., W. P. Hammes, M. Habermeyer, K. H. Engel, D. Knorr, and G.
Eisenbrand. 2011. Microbial food cultures. Opinion of the Senate Commission on Food Safety (SKLM) of the German Research Foundation (DFT). Molecular Nutrition & Food Research, 10.1002/mnfr.201100010, http://
www.dfg.de/download/pdf/dfg_im_profil/reden_stellungnahmen/2-1-/
sklm_microbial_food_cultures_100125_en.pdf.
2
General Tests and Assays
This Appendix addresses live microbial food cultures including probiotics (MFC) used as ingredients in foods. Whether
for food fermentation or probiotic use, viability of the MFC
is required at the time of use as a food ingredient. Depending on specific applications and processing conditions, such
as heat treatment of fermented foods, viability may not be
retained at the point of product consumption. In the case of
cultures used as probiotics, however, viability is essential at
the time of consumption. This Appendix is a broad description of this category of food ingredient and addresses, in a
general sense, the uses, enumeration methods, safety, regulatory status, identification, genetic and microbiological stability (throughout shelf life), purity, and product labeling of
MFC food ingredients.
MFC have been used in foods for millennia to transform a
variety of food substrates into fermented foods characterized
by their specific sensory properties (taste, aroma, texture,
color), improved stability, enhanced nutritive value, and perceived “wholesomeness” in general. Perishable foods such
as milk, vegetables, meat, fish, legumes, fruit, wine or beer
(for vinegar production), and cereals are converted into a
plethora of products that have a long tradition of food use
in all parts of the world. It is documented that between one
quarter and one third of the food consumed in Central Europe belongs to the category of fermented food.1 The microbes used to bring about these transformations are diverse
taxonomically and functionally, and include (but are not
limited to) species of the bacteria phyla Actinobacteria,
Firmicutes, and Proteobacteria as well as yeasts and filamentous fungi. Present knowledge about culture organisms is
essentially limited to those cultures that are commercially
available.
Currently MFC have two main roles in food processing or
production. The first role of MFC in foods is technological,
which refers to their role in food fermentation processes.
Generally speaking, in the case of MFC used strictly in fermentation processes, no health benefit claims are associated
with the presence of these microorganisms in the food. The
second role of MFC in foods is functional, which refers to
the perceived ability of certain defined live microbes to impart health benefits to the consumer. Health benefit claims
characterize this use and microbes in this second group are
called “probiotics.” According to an FAO/WHO Expert Consultation, probiotics are defined as “live microorganisms,
which when administered in adequate amounts confer a
health benefit on the host.”2 Often, the microbes useful for
technological transformations are not the same as those
used for healthful attributes. There are cases, however,
where one microbe can serve both purposes and a single
final food product can include MFC of both types. Typically,
the traits sought for probiotic functionality are different
from those sought for technological capacity. The understanding of the role of microbes in health and disease is
expanding rapidly3 and is likely to challenge underlying assumptions in this field as the knowledge base expands.
SAMPLE - FOR PROMOTIONAL USE ONLY
1478 / Appendix XV / General Tests and Assays
General Tests and Assays
Globally, many processes are still performed by spontaneous fermentation or with undefined cultures. The process
used is referred to as back shuffling or back slopping. Examples are found in the fermentative production of sauerkraut,
bread made from sourdough, vinegar, Italian parmesan
cheese, and a multitude of products in Asia and Africa. Numerous books and papers have been published describing
the great variety of microbial uses in foods.4,5,6,7
MFC Used as Probiotics Probiotics are live microorganisms which, when administered in adequate amounts, are
intended to confer a health benefit to the host.2 General
guidelines for their use in foods were advanced by a group
of FAO experts in 2002.8 Sanders discusses in detail what
probiotics are and, although this Appendix is limited to their
use in foods, notes that the term broadly encompasses multiple regulatory categories including foods, dietary supplements, and drugs (including genetically recombinant microbes).9 Probiotic use crosses species boundaries with their
use not only in humans, but, in this case as Microbial Feed
Cultures, in companion animals, animal agriculture, fish, and
birds.10
The use of probiotics in foods is motivated by the health
benefits purported to be conferred by them. Because of genetic, biochemical, and physiological differences among
strains of the same species, health benefits of probiotics are
considered to be specific to the strain (and intake levels)
tested.11 Any health benefits believed to be associated with
one strain of a probiotic (as a result of research trials) cannot be used to substantiate benefits of other strains of the
same species or genus without separate trials.
In food products, the claimed benefits of probiotics are
primarily focused on intestinal health and immune support.
These claims, however, are not entirely representative of the
full scope of human research conducted to date.12 Studies
have evaluated the role of probiotics in the immune system,
and in dietary management of patients with an ileoanal
pouch, infectious diarrhea in infants, enhanced gastrointestinal tolerance to antibiotic therapy, and lactose intolerance.9 Potential areas of emerging interest in probiotics in
Hui, Y. H. 2004. Handbook of Food and Beverage Fermentation Technology,
ed. Y. H. Hui. Vol. 134. Marcel Dekker.
6
Wood, B. J. B. 1998. Microbiology of Fermented Foods. Vol. 2. Blackie Academic & Professional.
7
Salminen, S., A. Wright, and A. Ouwehand. 2004. Lactic Acid Bacteria:
Microbiology and Functional Aspects. 2, revised ed. Marcel Dekker.
8
FAO. Guidelines for the Evaluation of Probiotics in Foods. In Food and
Agriculture Organization of the United Nations (FAO) [database online].
London, Canada, 2002 [cited 5 March 2011]. Available from ftp://ftp.fao.org/
es/esn/food/wgreport2.pdf.
9
Sanders, M. E. 2009. How Do We Know When Something Called “Probiotic” is Really a Probiotic? A Guideline for Consumers and Healthcare Professionals. Functional Food Rev: 3–12 (1), http://journals.bcdecker.com/pubs/FFR/
volume%2001,%202009/issue%2001,%20Spring/FFR_2009_00002/
FFR_2009_00002.pdf.
10
Giraffa, G., N. Chanishvili, and Y. Widyastuti. 2010. Importance of
Lactobacilli in Food and Feed Biotechnology. Research in Microbiology 161
(6:480–487) (Jul–Aug).
11
Sanders, M. E. 2007. Probiotics: Strains Matter. Functional Food and Nutraceuticals. June:34–41.
12
Sanders, M. E., G. R. Gibson, H. Gill, and F. Guarner. 2007. Probiotics in
Food: Their Potential to Impact Human Health. Council for Agricultural Science
and Technology, http://www.cast-science.org/publications/index.cfm/probiotics_their_potential_to_impact_human_health?show=product&productID=
2930.
5
FCC 9
the scientific community include some allergic diseases, irritable bowel syndrome, dental caries, respiratory infections,
Clostridium difficile toxin associated with antibiotic therapies,
and effects on absences or illness in the workplace or day
care center.13
Some of the ways that probiotics may impact the host:
• Probiotic production of antimicrobial substances, such as
organic acids or bacteriocins
• Probiotic cell components (ligands), which are recognized
by host immune and epithelial cells
• Probiotic delivery of functional proteins or enzymes (e.g.,
lactase)
• Up-regulation of immune response (e.g., secretory IgA)
• Down-regulation of inflammatory response
• Balancing immune responses
• Improvement of gut mucosal barrier function
• Enhancing stability or promotion of recovery of commensal
microbiota when perturbed
• Modulation of host gene expression
• Reduction of pathogen adhesion
• Modulation of gut microbiota population
Complicating the issue is the paucity of information on the
role of the food matrix on probiotic physiology and function.14 There is a growing trend toward the inclusion of
probiotics in a diversity of foods, including fermented dairy
products, dried breakfast cereals, juices, and nutrition bars.
Maintaining viability of the probiotic in these different food
formulations and their concomitant storage conditions can
be challenging, and it may be more difficult to understand
if or how these different food products impact the physiological function of the probiotic in vivo.
MFC Production The MFC defined in this Appendix are
produced in specialized fermenters or cabinets under strict
hygiene conditions. The typical fermentation process for
MFC is described in this section.
The original microbial strains are stored in a microbiology
laboratory, where a small quantity of microbes—the inoculation material—is prepared to start the production process
for every batch. This inoculation material is transferred to
fermenters for liquid fermentation or to a solid medium for
surface growth. Media are tailored to the specific requirements of the microbial species and typically contain proteins, carbohydrates, vitamins, and minerals. The culture is
allowed to multiply and grow under carefully defined and
monitored conditions.
After the microbial cells are grown, the cultured cells are
harvested, often by centrifugation, and the biomass is preserved in liquid, frozen, or powder form. This substance
then undergoes final formulation, which may involve blending of multiple cultures, prior to shipment to the food
manufacturer.
13
Douglas, L., and M. E. Sanders. 2008. Probiotics and Prebiotics in Dietetic
Practice. J Amer Dietetics Assoc. 108: 510–521.
14
Sanders, M. E., and M. L. Marco. 2010. Food Formats for Effective Delivery of Probiotics. Annual Review of Food Science and Technology 1 (1:65–85)
(04/01; 2011/02).
Copyright United States Pharmacopeia, 2014. All rights reserved.
SAMPLE - FOR PROMOTIONAL USE ONLY
FCC 9
The most developed area of MFC application is their use
as starter cultures in dairy products. Lactic acid bacteria contained therein are provided as single-strain, multi-strain or
multi-strain preparations.15
Starter culture bacteria in dairy products, meat, wine,
sourdough, and other traditional fermented foods contribute
to the common properties of these products. In each case,
the starter culture bacteria fulfill product-specific tasks such
as coagulation of casein, production of texture-enhancing
exopolysaccharides, formation or prevention of gas or hydrocolloids in dairy products, improved sliceability, enhanced reddening in meat, malolactic fermentation in wine,
or ensured bakability in sourdough used for rye bread.
The historic source of these cultures is typically microbes
derived from natural, spontaneous fermenting food substrates such as fermenting cream, milk or cheese. The starter
cultures and fermentation conditions are specific for each
dairy product. For example, the thermophilic microorganisms Lactobacillus delbrueckii subspec. bulgaricus and Streptococcus thermophilus are used to produce the fermented food
known as “yogurt”, while the mesophilic microorganisms
Lactococcus lactis subspec. cremoris and subspec. diacetylactis, Leuconostoc mesenteroides subspec. cremoris and Leuconostoc lactis are used for production of sour milk, sour
cream and cheese.
GLOBAL REGULATORY STATUS
IDENTITY
Defined starter cultures used for their technological effect
may be identified at the genus, species, or strain level depending on the requirements of their use. The purity of
starter cultures is determined by the level or absence of undesired microorganisms. Undesired microorganisms include
pathogens, the standard indicator organisms for pathogens,
and microbes that negatively impact technical properties,
such as spoilage yeasts and molds in yogurt cultures. The
trueness-to-type of defined starter cultures is assured by using biochemical or molecular techniques to identify the
strains that are present.
Proper identification of a probiotic to the genus, species,
and strain level is an important component of the FAO/
WHO guidelines for use of probiotics in food.8 This information is needed to evaluate the safety of a microbe for use as
a probiotic. A means of identifying the specific strain when
combined with other microbes in the product and, ideally,
of identifying the specific strain from the mixed microbial
communities present in a human host is also recommended.
15
Mayra-Makinen, A., and M. Bigret. 1998. Industrial Use and Production of
Lactic Acid Bacteria. In Lactic Acid Bacteria: Microbiological and Functional Aspects., eds. S. Salminen, A. Wright. 1st ed. Vol.: 73–102. Marcel Dekker.
Copyright United States Pharmacopeia, 2014. All rights reserved.
Strain-specific isolation from humans who have consumed
the strain is helpful during studies launched for efficacy testing and is critical if there is some suspicion of adverse
events due to consumption of the strain. Proper identification to the strain level is needed to achieve correct packaging labeling. Typically, DNA-based methods, as described
below under Taxonomy—Identification, are the most reliable
for identification. It is becoming more common for whole
genomic sequences to be known for commercial probiotic
strains.16
Bacterial Starter Culture Bacterial starter cultures that
contain one or several strains of microorganisms at high
counts (in general more than 108 colony forming units of
viable bacteria per gram or mL, CFU/g or CFU/mL) are
added to the fermentation substrate and bring about desirable metabolic reactions (e.g., fermentation of lactose resulting in acid production, degradation of lactic acid to propionic acid, reduction of nitrate to nitrite reacting further to
nitrosomyoglobin in meat) or other metabolic activities directly related to specific product properties.
Examples of bacteria of importance for the food industry17
(not an inclusive list):
• Arthrobacter
• Bifidobacterium
• Brevibacterium
• Corynebacterium
• Enterococcus
• Hafnia
• Halomonas
• Kocuria
• Lactobacillus
• Lactococcus
• Leuconostoc
• Micrococcus
• Oenococcus
• Pediococcus
• Propionibacterium
• Staphylococcus
• Streptococcus
Yeast Starter Culture Yeast starter cultures that contain
one or several strains of yeast at high counts (in general
more than 108 CFU/g or CFU/mL of viable cells) are added
to bring about a desirable metabolic reaction (e.g., fermentation of carbohydrates into carbon dioxide or ethanol) or
other metabolic activities directly related to specific product
properties.
Examples of yeasts of importance for the food industry17,18
(not an inclusive list):
• Debaryomyces
• Galactomyces
Ventura, M., S. O’Flaherty, M. J. Claesson, F. Turroni, T. R. Klaenhammer,
D. van Sinderen, and P. W. O’Toole. 2009. Genome-Scale Analyses of HealthPromoting Bacteria: Probiogenomics. Nat Rev Microbiol. 1 (7).
17
Morgensen, G., S. Salminen, J. O’Brien, A. Ouwehand, W. Holzapfel, C.
Shortt, R. Fonden, et al. 2002:2. Inventory of Microorganisms With a Documented History of Use in Food. Bulletin of the international Dairy Federation:
10–19 (01/01).
18
EFSA Panel on Biological Hazards. Scientific Opinion on the Maintenance
of the List of QPS Biological Agents Intentionally Added to Food and Feed
(EFSA Scientific Committee Table 1: Assessment of Yeasts with Respect to a
Qualified Presumption of Safety) (2010 Update). 2010 [cited 5 March 2011].
Available from http://www.efsa.europa.eu/en/efsajournalpub/1944.htm.
16
General Tests and Assays
Species used as MFC that have a long history of safe use are
considered to be traditional food ingredients and are legally
permitted for use in human food all over the world. As national food law changes frequently, specific country regulations are not covered here. Producers should refer to the
relevant food law of the country of interest for specific regulatory information.
General Tests and Assays / Appendix XV / 1479
SAMPLE - FOR PROMOTIONAL USE ONLY
1480 / Appendix XV / General Tests and Assays
•
•
•
•
•
•
•
•
•
•
•
•
•
Hanseniaspora
Hansenula
Isaatchenkia
Kluyveromyces
Metschnikowia
Pichia
Saccharomyces
Schizosaccharomyces
Torulaspora
Yarrowia
Willopsis
Zygosaccharomyces
Xanthophyllomyces
General Tests and Assays
Fungal Starter Culture Fungal starter culture that contains one or several strains of fungi at high counts (in general more than 108 CFU/g or CFU/mL) are added to bring
about desirable metabolic activities (e.g., proteolysis, lipolysis, transformation of flavor precursors, taste, flavor, color
and ripening) or other activities directly related to specific
product properties.
Examples of fungi of importance for the food industry17
(not an inclusive list):
• Geotrichum
• Penicillium
• Aspergillus
Taxonomy—Identification Taxonomy is a discipline in
biology that describes the relationship between organisms
and captures it in a hierarchical system: phyllum, class, order, family, genus, species, and strain.
An MFC preparation may be composed of one or several
genera, species, or strains. In the field of microbiology, a
species is a population of strains in which all share the same
major properties but differ from other collections of strains
in one or more significant properties.
For microorganisms, this determination has historically
been based on the examination of phenotypic (e.g., cell
morphology) or physiological characteristics (e.g., sugar fermentation), or chemotaxonomic properties (e.g., guaninecytosine content). More recently, molecular biological methods based on genetic examination have been routinely applied, clearly reflecting the phylogenetic relationship between species. The most common of these methods are
DNA/DNA-hybridization and 16S rDNA sequencing. The International Committee on Systematics of Prokaryotes (ICSP)
uses high quality full-length 16S rDNA sequencing as their
method for differentiating species. According to ICSP, populations of strains sharing at least 97% similarity of 16S rDNA
gene sequences and exhibiting >70% DNA/DNA-hybridization are considered to belong to the same species.19,20
[NOTE—Guidance for taxonomic identification of bacteria
and yeast, including explanations of state-of-the-art identification techniques, can be found in the latest Edition of Bergey’s Manual of Systematic Bacteriology (available from the
19
Logan, N. A., O. Berge, A. H. Bishop, H.-J. Busse, P. DeVos, D. Fritze, M.
Heyndricks, P. Kampfer, L. Rabinovitch, M. S. Salkinoja-Salonen, L. Seldin, and
A. Ventosa. (2009). Proposed minimal standards for describing new taxa of
aerobic, endospore-forming bacteria. Int J Syst Evol Microbiol. 59, 2114–2121.
20
Tindall, B. J., R. Rossello-Mora, H.-J. Busse, W. Ludwig, and P. Kampfer.
(2010). Notes on the characterization of prokaryote strains for taxonomic
purposes. Int J Syst Evol Microbiol. 60, 249–266.
FCC 9
Bergey’s Manual Trust, www.bergeys.org). Information
about the current classification of fungi can be obtained
from the International Commission on the Taxonomy of
Fungi (ICTF, www.fungaltaxonomy.org).]
QUANTIFYING NUMBERS AND
ACTIVITIES OF MFC
Enumeration of MFC cannot be generalized because of the
broad range of microbes encompassed by MFC, and the
resulting large differences in methodology required to accommodate the differing physiology and growth
characteristics.
Defined starter cultures used for their technological effect
are usually standardized on activity or vitality, which is more
commercially important than cell count. Sausage cultures,
for example, are standardized on their acidifying activity,
while bakers’ yeast cultures are standardized on their leavening power.
The time required for acidification to reach a certain pH
value (in short, called “time to pH”) is used by many as a
guideline for the activity of cultures. A specific quantity of
culture is inoculated in a specified amount of standardized
substrate (e.g., milk) and subsequently incubated at a constant temperature for a given time. During incubation the
acidification activity is measured by continued pH measurements using a pH electrode. “Time to pH” is specified in
minutes and a higher value indicates a longer time to reach
the given pH value.
Probiotics typically rely on a count of live microbes as a
measure of activity. Standard methods for enumeration of
bacteria are publicly available,21 covering Bifidobacterium,
Lactobacillus, Lactococcus, Leuconostoc, Pediococcus, Propionibacterium, Streptococcus and others. Yeast and fungi have
other variants of enumeration standards. Each manufacturer
can and typically does adopt one recommended method for
its particular strain collection. For the appropriate enumeration of a strain or several strains in a specific commercial
blend, the manufacturer is the best source of information
regarding enumeration methods.
SAFETY
[NOTE—This section refers to the safety of MFC products
under intended use and does not include safe handling information, which should be procured from manufacturers or
suppliers of MFC.]
Presumption of safety of a microbe may be based at the
genus, species, or strain level, depending on the characteristics of the microbe and the end use.
21
ISO 27205:2010(E) / IDF 149:2010(E) Annex A (IDF 149 2010), available
from the International Organization for Standardization at http://
www.iso.org.
Copyright United States Pharmacopeia, 2014. All rights reserved.
SAMPLE - FOR PROMOTIONAL USE ONLY
FCC 9
MFC are covered by general food laws. They must all
comply with these fundamental provisions, meaning that
they must be considered safe and suitable for their intended
use. In order to document microorganisms traditionally used
as food ingredients, the International Dairy Federation
(IDF)22 in collaboration with the European Food and Feed
Cultures Association (EFFCA)23 has compiled a nonexhaustive
inventory of microorganisms with a documented history of
use in food. An updated list is currently elaborated. No exhaustive list exists at this time.
The safety of probiotics is presumed for certain species if
there is a properly documented history of safe use. Probiotics, especially from the Lactobacillus and Bifidobacterium species, when used in foods for the generally healthy population, are reported to be safe.24,25 Strain-specific
characterization of safety is required, however, to address
the issue of the presence of transferable antibiotic resistance
traits.
22
Morgensen, G., S. Salminen, J. O’Brien, A. Ouwehand, W. Holzapfel, C.
Shortt, R. Fonden, et al. 2002:1. Food Microorganisms - Health Benefits,
Safety Evaluation and Strains with a Documented History of Use in Food.
Bulletin of the International Dairy Federation: 4–9 (01/01).
23
EFFCA. IDF-EFFCA Inventory of Microorganisms. 2002 [cited 5 March
2011]. Available from http://www.effca.org/views.html.
24
Borriello, S. P., W. P. Hammes, W. Holzapfel, P. Marteau, J. Schrezenmeir,
M. Vaara, and V. Valtonen. 2003. Safety of Probiotics That Contain Lactobacilli
or Bifidobacteria. Clin Infect Dis. 35:775–780.
25
Sanders, M. E., L. M. Akkermans, D. Haller, C. Hammerman, J. Heimbach,
G. Hormannsperger, G. Huys, et al. 2010. Safety Assessment of Probiotics for
Human Use. Gut Microbes 1 (3:164–185) (May).
26
CFR. Code of Federal Regulations title 21 CFR 170.3(i) Food and Drugs
Chapter I—Food and Drug Administration Department of Health and Human
Services subchapter B—Food for Human Consumption. 2007 [cited 1 April
2011]. Available from http://www.accessdata.fda.gov/scripts/cdrh/cfdocs/cfcfr
/CFRSearch.cfm?fr=170.3.
Copyright United States Pharmacopeia, 2014. All rights reserved.
Qualified Presumption of Safety by the European
Food Safety Authority The Qualified Presumption of
Safety (QPS) system, started in November 2007,27 was implemented to replace the case-by-case safety assessment of
microorganisms introduced in the food chain with a streamlined, harmonized procedure. It applies when evaluating microorganisms that require a market authorization (e.g., feed
cultures, cell factories producing enzymes, plant protection,
and others), some of which may be used in food as well.
The QPS assessment is based on the taxonomic level, the
body of knowledge, the history of use, and identification of
potential safety concerns. The assessments result in a list of
microorganisms at the species level that are presumed safe
for use, independent of media, fermentation conditions, and
intended use. Food cultures with a long history of safe use
are considered to be traditional food ingredients and are
legally permitted for use in human food in the EU without
pre-market authorization. QPS can, however, be a practical
guide to be used by manufacturers of microbial food cultures, including probiotics, to specifically and systematically
evaluate the safety of a new strain before it is marketed.
It is common practice to screen MFC for abnormal or
transferable antibiotic resistance. Such tests have been reviewed and published for milk and milk products.28 Subsequent tests by industry on multiple food and supplement
products have shown similar results as outlined in the document referenced. The methods in the document referenced
are, therefore, widely accepted and recognized.
LABELING & POTENCY (END OF SHELF
LIFE VIABILITY)
Labeling shall be in accordance with national legislation.
Please refer to the country of interest for further
information.
Defined starter cultures used for their technological effect
are commonly labeled consistent with their identity and activity. Dairy starter cultures, for example, are sold with instructions for the applications and dose rates where they are
to be used.
Each genus and species designation, consistent with the
most current scientifically recognized nomenclature, should
be listed on the label along with potency (typically expressed as CFU/g or CFU/serving) or a measure of the acidification activity (where applicable). For probiotic applications, the strain designation should also be listed on the
label for each strain. Use of a trademarked name does not
replace the need for the label to include the correct genus,
species, and strain designation.
27
EFSA. Introduction of a Qualified Presumption of Safety (QPS) Approach
for Assessment of Selected Microorganisms Referred to EFSA—Opinion of the
Scientific Committee. 2007 [cited 5 March 2011]. Available from http://
www.efsa.europa.eu/en/topics/topic/qps.htm.
28
ISO 10932:2010 (IDF 223:2010). Milk and Milk Products—Determination of
the Minimal Inhibitory Concentration (MIC) of Antibiotics Applicable to
Bifidobacteria and non-Enterococcal Lactic Acid Bacteria (LAB). ISO copyright
office, Case Postale 56, CH-1211 Geneva 20: International Organization for
Standardization, 2010.
General Tests and Assays
U.S. GRAS Approach According to the current U.S. federal law and regulations, a substance (other than a color
additive) that, under the conditions of its intended use, is
“generally recognized as safe” (GRAS) by experts who are
qualified by scientific training and experience in the evaluation of the safety of foods and food ingredients, may be
used in food for that purpose without pre-market approval
from the Food and Drug Administration (FDA). A determination that the use of a substance is GRAS (i.e., a GRAS determination) may be based either on common use in foods
prior to 1958 or on scientific procedures. For purposes of a
GRAS determination, the term “safe” means that there is
reasonable certainty in the minds of competent scientists
that a substance is not harmful under intended conditions
of use.26 Because safety is assessed in the context of “intended use,” a microorganism that is GRAS for certain uses
may not be GRAS for other uses (e.g., incorporation in other
categories of foods, addition at higher concentrations).
General recognition of safety based upon scientific procedures calls for the same quantity and quality of scientific
evidence as would be required to obtain an FDA approval of
a food additive. “Scientific procedures” include human,
animal, analytical, and other studies, whether published or
unpublished, appropriate to establish the safety of a substance. The pivotal data supporting a GRAS determination
based on scientific procedures must be publicly available,
whereas corroborating data need not be public.
General Tests and Assays / Appendix XV / 1481
General Tests and Assays
SAMPLE - FOR PROMOTIONAL USE ONLY
1482 / Appendix XV / General Tests and Assays
FCC 9
VIABILITY AND GENETIC STABILITY
and the product. For lactic acid bacteria, there are public
standards that may be used to establish control measures.29
For other MFC, test criteria have to be relevant depending
on the genus, species, and application of the starter culture.
The manufacturer should establish measures to control
any potential cross-contamination from other products that
might affect the quality of the product.
Shelf life viability is both strain and food application specific.
Therefore, the manufacturer of the MFC should be contacted for details on the shelf life viability of its product. It is
also imperative, however, to verify the viability of the MFC
in the final food matrix. The viability of the organism over
the course of shelf life in the final food matrix may vary
greatly, and must be substantiated by the food producer. In
some cases MFC do not retain viability in the final fermented food (e.g., sour dough bread or pasteurized sauerkraut), rendering the final product shelf life viability assessment irrelevant. In the case of a probiotic-containing food,
product labels should provide an indication of the levels of
viable probiotic through the end of the shelf life, and this
level should correspond to a level found to promote any
claimed health benefits that have been determined from
human studies.
Genetic stability is also strain-specific and the manufacturer of the live MFC should be contacted for those details.
The following measures are good practices to help maintain
long-term genetic stability and avoid genetic drift:
• A centralized in-house strain bank should be responsible for
the supply of each strain to all production sites. This can
prevent deviations in the strains at each production site.
• Systems should be in place for comprehensive documentation and traceability of each strain.
• A long-term storage plan that minimizes the number of
generation times should be implemented.
• DNA-fingerprinting of new batches and comparison to previous batches should be routine. [NOTE—Use of state-ofthe-art genetic techniques is essential.]
• Basic phenotypic characteristics of new batches should be
verified.
PURITY
To control the essential composition of starter cultures, the
manufacturer should put in place, implement and maintain
a quality management system and a permanent procedure
or procedures based on prerequisite programs and hazard
analysis critical control point (HACCP) or similar principles.
This should include a rigorous quality control process ensuring that no substandard batches of culture are produced. In
addition to regularly checking the equipment, the quality
control process should also include confirming the sterility
of the production material, characterizing and monitoring
the bacterial strains that are used to prepare the inoculation
material, as well as monitoring the characteristics of the
product at various stages of production. Parameters should
be continuously checked by automatic sensors, and the process data should be stored. The microbiological condition
should be monitored to detect any external (or foreign)
contamination to avoid cross-contamination from within the
fermentation. If any contamination is detected, the production process should be stopped immediately and not restarted until the problem has been solved.
Control measures for preventing potential contamination
should be established. The microbiological criteria and specifications for process hygiene and food safety criteria should
be set in order to define the acceptability of the processes
INTERNATIONAL DEPOSIT / REFERENCE
STRAIN MATERIAL
There is no requirement for the deposit of MFC used as
fermentation agents in international culture collections. The
strain-specificity of purported probiotic effects and the importance of human research studies used to document
health benefits attributed to microorganisms, however, illustrate the importance of depositing strains used as probiotics
in international culture collections. The deposited material
provides a reference material for future use of the probiotic
and facilitates accessibility of the strain for confirmatory efficacy research.
One culture collection where strains may be deposited is
the American Type Culture Collection (ATCC). ATCC is an
independent, private, nonprofit biological resource center
(BRC) and research organization. ATCC authenticates microorganisms and cell lines and manages the long-term preservation of the cultures for the scientific community. ATCC
also characterizes biological products (including cell lines,
fungi, protozoa, bacteria, and viruses) and develops and
evaluates biological assays along with evaluating testing
methodologies. Another culture collection is maintained by
the German Collection of Microorganisms and Cell Cultures
(DSMZ). DSMZ is a leading BRC in Europe and works in a
manner similar to ATCC in collecting and maintaining microorganisms and cell lines along with other types of biological materials for use by the scientific community. Like
ATCC, DSMZ is an independent and nonprofit organization.
PROCEDURES FOR MAINTAINING
STRAINS (MOTHER CULTURES)
The following are generally considered to be practices which
will help maintain mother culture purity and identity:
• Identification of the strain species by well-acknowledged
and state-of-the-art techniques (including 16S rDNA for
bacteria, 18S rDNA for yeasts and molds)
• Production of mother stock cultures under strict, hygienic
conditions
• Storage below −80°
• Adherence to rigorous hygiene control procedures to exclude any contamination with foreign microorganisms and
cross-contaminants
• Verification of strain purity
• DNA-fingerprinting to follow genetic stability
• Examination of phenotypic properties
• Use of a storage regimen that minimizes the number of
strain transfers
IDF 149. Fermented Milk Products—Bacterial Starter Cultures—Standard of
Identity. ISO Copyright Office, Case Postale 56, CH-1211 Geneva 20: International Organization for Standardization, 2010.
29
Copyright United States Pharmacopeia, 2014. All rights reserved.
SAMPLE - FOR PROMOTIONAL USE ONLY
FCC 9
SHIPPING AND STORAGE
Shipping and storage conditions will necessarily be adapted
to the particular strains being sold. The manufacturer should
General Tests and Assays / Appendix XV / 1483
be contacted for details on proper shipping and storage of
its culture product. Furthermore, storage instructions are
generally described on the product label and should be
closely followed.
General Tests and Assays
Copyright United States Pharmacopeia, 2014. All rights reserved.