最近的一次作业——翻译（blogcn: 2006-10-18 22:12）

12
Probiotic functional foods
T. Mattila-Sandholm and M. Saarela, VTT Biotechnology, Espoo

12.1 Introduction: the health benefits of probiotic foods
The area of food for health has been identified as a priority area for research in
Europe. This is based on the recognition that there is enormous potential for
improving health through food. Furthermore, diet is a major focus of public
health strategy aimed at maintaining optimum health throughout life, preventing
early onset of chronic diseases such as gastrointestinal disorders, cardiovascular
disease, cancer and osteoporosis, as well as promoting healthier ageing.
Although the highly complex relationship between food and health is still poorly
understood, recent research advances in different disciplines provide promising
new approaches to improve our understanding. The growing demand for
‘healthy’ foods is stimulating innovation and new product development in the
food industry internationally. Indeed, the food industry has a central role in
facilitating improved eating practices through the provision and promotion of
healthy foods.

Probiotics are live microbial food supplements which benefit the health of
consumers by maintaining or improving their intestinal microbial balance.1 Due
to their perceived health benefits probiotic bacteria have been increasingly
included in yoghurts and fermented milks during the past two decades. Most
commonly they have been lactobacilli such as Lactobacillus acidophilus, and
bifidobacteria often referred to as ‘bifidus’ (see Table 12.1).2 A major
development in functional foods pertains to foods containing probiotics and
prebiotics which enhance health-promoting microbial flora in the intestine.
There is growing scientific evidence to support the concept that the maintenance
of healthy gut microflora may provide protection against gastrointestinal
disorders including gastrointestinal infections, inflammatory bowel diseases and
even cancer. The use of probiotic bacterial cultures stimulates the growth of
preferred micro-organisms, crowds out potentially harmful bacteria and
reinforces the body’s natural defence mechanisms.

Before a probiotic can benefit human health it must fulfil several criteria: it
must have good technological properties so that it can be manufactured and
incorporated into food products without losing viability and functionality or
creating unpleasant flavours or textures; it must survive passage through the
upper gastrointestinal tract and arrive alive at its site of action; and it must be
able to function in the gut environment. To study the probiotic strain in the
gastrointestinal (GI) tract, molecular techniques must be established for
distinguishing the ingested probiotic strain from the potentially thousands of
other bacterial strains that make up the gastrointestinal ecosystem. Techniques
are also required to establish the effect of the probiotic strain on other members
of the intestinal microbiota and importantly on the host. This includes not only
positive health benefits, but also demonstration that probiotic strains do not have
any deleterious effects. Armed with this knowledge, the probiotics can then
enter human clinical pilot studies that attempt to assess their clinical health
benefits to consumers (Table 12.2).3, 4

12.1.1 Demonstration of Nutritional Functionality of Probiotic Foods
(FAIR CT96-1028)
Europe has traditionally had a leading position on the probiotic market.
Considerable confusion and scepticism, however, exists on the side of consumers,
consumer organisations and certain quarters of the scientific community about the
claims associated with probiotic products. This greatly hampers further
exploitation of functional foods containing probiotic bacteria and weakens the
market position of European producers in the face of competition. To eliminate
these hurdles, to speed up adaptation of the probiotic food technology and to
enhance the attractiveness of new probiotic foods, it is essential to demonstrate
the up-to-date basis for marketable claims by presenting the health and nutritional
benefits of probiotic bacteria and foods. Special emphasis should be put on
intestinal integrity and immune modulation, exploitation of validated methods for
the selection of novel probiotic bacteria and foods, and dissemination of the
obtained knowledge to the extended audiences consisting of industries,
authorities and consumers. The Probdemo project was initiated to demonstrate
the value of probiotic products to European consumers. The project objectives
were divided into four interactive tasks (Table 12.3):

1. To establish a scientifically based selection of probiotic bacterial strains
currently available for functional foods. Six probiotic strains representing
Lactobacillus and Bifidobacterium species were chosen for demonstration
purposes.
2. To demonstrate the beneficial value of probiotic products in human pilot
trials both in children and adults. Initial tests showed that probiotic strains
did not have any deleterious effects in healthy children or adults.
Furthermore, probiotic strains were shown to be effective in the treatment
of infants with food allergy and small children with rotavirus diarrhoea. The
effect of probiotics was also demonstrated in adults with inflammatory
bowel disease (IBD).
3. To demonstrate and meet the functional and technological requirements
essential for the industrial production of probiotics as functional foods. This
has been established by studying probiotic strain properties in vitro and
reflecting these results to the clinical situations. The main focus has been on
demonstrating adhesion in vitro and in vivo using human biopsies, on
demonstrating the technological criteria for probiotic products, and on pilot
production of probiotic strains.
4. To disseminate the knowledge and results to extended audiences consisting
of industrial users, authorities and consumer organisations. This has been
established by annual workshops (Workshop 1 was held on Safety of
Probiotics in 1996, Workshop 2 on Probiotic Research Tools in 1997,
Workshop 3 on Functional Food Research in 1998, Workshop 4 on
Functional Foods in 2000).5–8

The project participants and institutes collectively have wide experience in
this research area, building on the results of former EU programmes on lactic
acid bacteria and probiotics. The industrial partners have long traditions in the
markets of functional foods with special reference on probiotic products. VTT
Biotechnology, Finland, has the role of coordination and dissemination of
activities and demonstration tasks on probiotic strain properties, technological
properties and clinical testing on adults. The University of Wageningen,
Netherlands, has the key role of demonstrating the activity and viability of
probiotic strains in human clinical trials by using molecular methods including
PCR, in situ hybridisation and DGGE/TGGE. The Catholic University of
Piacenza, Italy, has the role of showing the adhesive and aggregation properties
of the strains to be demonstrated. The University of College Cork, Ireland, has
profound expertise on human gastroenterology, clinical testing with adults and
immune modulation activities of probiotics. University of Turku, Finland, has a
long scientific tradition of clinical testing with children and the links between
clinical pediatrics and functional foods. The industrial partners Valio Ltd.
(Finland), Arla (Sweden), Nestle´ (Switzerland) and Christian Hansen Laboratories
(Denmark) have sound basis of industrial production of probiotic products
and long experience on functional foods market as well as research in this area.
This industrial role is of utmost importance in selecting the strains to be
demonstrated, preparing the products to be developed and in verifying their
beneficial effects.

12.2 Selecting probiotic strains
The theoretical basis for selection of probiotic micro-organisms illustrated in
Table 12.4 and Fig. 12.1 includes:
• safety
• functional behaviour (survival, adherence, colonisation, anti-microbial
production, immune stimulation, anti-genotoxic activity and prevention of
pathogens such as Helicobacter pylori, Salmonella, Listeria and Clostridium)
• technological aspects (growth in milk, sensory properties, stability, phage
resistance, viability in processes).

In general, strains for pilot testing should be selected based on established in
vitro scientific data. Naturally, the safety of probiotic strains has been of prime
importance and new guidelines have been developed.9–13 Current safety criteria
and functional properties for successful probiotics have been defined in recent
reviews.14–16 These include the following specifications:
• Strains for human use are preferably of human origin.
• They are isolated from healthy human GI tract.
• They have a history of being non-pathogenic even in immunocompromised
hosts.
• They have no history of association with diseases such as infective
endocarditis or GI disorders.

The significance of human origin has been debated recently, but most if not
all current successful strains are indicated to be of human origin. Similarly, the
importance of the ability to colonise the human gastrointestinal tract has been
questioned. However, most current strains are reported to persist in humans at
least temporarily as measured by faecal counts following ingestion. Acid and
bile stability are self-evident properties for any strain expected to have effects in
the intestinal tract. Ability to adhere and persist are also closely related to
potential immune effects. It is likely that some mechanisms of adhering and/or
binding to the intestinal cells are required. Thus controlled comparable studies
on in vitro model systems, such as the Caco-2 cell line, are of importance.17, 18
Adherent strains of probiotic bacteria are favoured since they are likely to persist
longer in the intestinal tract and thus have better possibilities of showing
metabolic effects than non-adhering strains. At least one of the commercial
probiotic strains has been demonstrated to adhere to the colonic mucosae in
vivo.19

To have an impact on colon flora it is important for probiotic strains to show
antagonism against pathogenic bacteria via anti-microbial substance production
or competitive exclusion. Enormous research efforts have focused on bacteriocin
research. However, the mode of action and efficacy of bacteriocins in the gut is
not known for probiotic bacteria. Although probiotic strains may produce
can only be limited, since traditional bacteriocins have an inhibitory effect only
against closely related species such as other Lactobacillus or on sporeformers
such as Bacillus or Clostridium. However, low molecular weight metabolites
(and secondary metabolites) may be more important since they show wide
inhibitory spectrum against many harmful organisms like Salmonella,
Escherichia coli, Clostridium and Helicobacter.20–22

12.2.1 Development of cultures aimed for functional probiotic foods
Probiotic dairy foods and cultures have a long history and large consumption in
the Nordic diet. Industrial products, including cultured dairy products, and their
probiotic properties have been studied for many decades. The objective of the
Nordic programme (from 1994 to end of 1997) was to validate industrial
probiotic strains with regard to in vitro functionality. Strains were provided by
project participants Arla (Sweden), Christian Hansen (Denmark), Norwegian
Dairies (Norway) and Valio Ltd (Finland). Strains studied included the
following: Lactobacillus paracasei subsp. paracasei strains E-94506 and E-
94510, Lactobacillus rhamnosus strains E-94509 and E-94522, Lactobacillus
acidophilus E-94507, Lactobacillus plantarum E-79098, Lactococcus lactis
subsp. lactis E-90414, L. lactis subsp. cremoris E-94523, Bifidobacterium
animalis (lactis) E-94508 and Bifidobacterium longum E-94505. The studies
included research on the in vitro cytokine release effects, adhesive properties,
anti-mutagenicity and behaviour in the gastrointestinal tract models (TNO
gastrointestinal tract model in the Netherlands, and the SHIME ecosystem at
Ghent, Belgium). Also technological and production properties were assessed.

Assessment of adhesion properties
Adhesion of probiotic strains to human intestinal cells and the following
colonisation of the human gastrointestinal tract has been suggested as an
important prerequisite for probiotic action. Adhesion verifies the potential of the
strain to inhabit the intestinal tract and to grow in intestinal conditions. Adhesion
also provides an interaction with the mucosal surface facilitating contact with
gut-associated lymphoid tissue mediating local and systemic immune effects.
Thus, only adherent probiotics have been thought to induce immune effects and
to stabilise intestinal mucosal barrier.23 The Nordic programme project results of
in vitro adhesion assays gave a clear indication of differences and variation
between assays and different strains.24, 25 It was evident that in addition to Caco-
2 cell line experiments, other test systems were also needed to characterise the
adhesion potential and different adhesion mechanisms. The adhesion system was
also used to study the anti-invasion potential of probiotic stains. Different
probiotic strains show relatively different behaviour in invasion inhibition and
novel methodologies are needed to assess these properties in a way that relates
them to clinical situations. Adhesion experiments indicate clear differences in
the colonisation potential of different probiotic strains and, when later connected
with clinical data, may provide a useful basis for selection and method
development for future probiotic strains.17, 18 Lately adhesion assays have also
been applicated to human ileostomy glycoproteins (modelling for small
intestinal mucus), showing once again different characteristics of the probiotic
features.26

In vivo adhesion studies using colonic biopsies
Faecal samples have been used in most colonisation studies on probiotic
bacteria.27, 28 These, however, reflect only the bacteriological situation in faecal
material and do not give an accurate picture about the situation in different parts
of the gastrointestinal tract or in the mucosal layer of the gut. There are
advantages in taking biopsy material from colonoscopy patients: in this way
tissue samples have been obtained, not only from the rectal-sigmoidal region,
but also from other parts of large intestine (ascending, transverse and descending
colon). As a result the preferential adhesion of a commercial probiotic strain
(Lactobacillus GG) to the descending part of large colon was detected by using
biopsy material. This probiotic strain was shown to survive in the gut epithelium
for several days after consumption of the probiotic preparation was stopped and
even after the strain could no longer be detected in faecal samples.19 Johansson
and co-workers have also demonstrated the adhesion of different Lactobacillus
strains to rectal mucosal biopsy samples obtained from volunteers who had
consumed fermented oatmeal soup.29

Immunological assessment
Gut-associated lymphoid tissue may have contact with adhesive probiotic
preparations and therefore adhesion is one way of provoking immune effects.
The Nordic network studied the interactions of probiotic strains and dairy
cultures (Lactobacillus bulgaricus, Streptococcus thermophilus) with cytokine
production (human TNF-, interleukin-6, interleukin-10, interleukin-12, TGF-,
and interferon-). Probiotic strains which had passed through the in vitro TNO
gastrointestinal tract model were also assayed for their ability to induce cytokine
production (TNF-, interleukin-6).30 The main goal was to investigate whether
probiotic strains stimulate the immune system in vitro through cytokines. IL-6
production showed considerable variation between experiments performed with
live bacteria. Test strains were not observed to induce IL-10.31 Efforts were
made to develop new methods to measure early cytokine responses by detection
of mRNA by Northern hybridisation. This method proved more sensitive than
the ELISA and has demonstrated that probiotic strains indeed produce IL-10 and
IL-1b. Further investigations focused on analysis of the pathway of cytokine
induction by probiotics, estimation of the effect of serum proteins, interaction
between probiotics and human cell surface molecules.32