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Research in Microbiology 161 (2010) 480 e 487 www.elsevier.com/locate/resmic
Research in Microbiology 161 (2010) 480 e 487 www.elsevier.com/locate/resmic

Research in Microbiology 161 (2010) 480 e487

Research in Microbiology 161 (2010) 480 e 487 www.elsevier.com/locate/resmic

Importance of lactobacilli in food and feed biotechnology

Giorgio Giraffa a , * , Nina Chanishvili b , Yantyati Widyastuti c

a Agricultural Research Council, Research Centre for Forage and Dairy Productions (CRA-FLC), Via Lombardo 11, 26900 Lodi, Italy b The G. Eliava Institute of Bacteriophage, Microbiology and Virology, 0160 Tbilisi, Georgia c Research Center for Biotechnology, Indonesian Institute of Sciences, 16911 Cibinong, Indonesia

Received 14 October 2009; accepted 2 March 2010 Available online 17 March 2010

Abstract

The genus Lactobacillus is a heterogeneous group of lactic acid bacteria (LAB) with important implications in food fermentation. The ability to colonize a variety of habitats is a direct consequence of the wide metabolic versatility of this group of LAB. Consequently, lactobacilli have been used for decades in food preservation, as starters for dairy products, fermented vegetables, fish and sausages as well as silage inoculants. Lactobacilli have also been proposed as probiotics and microbial cell factories for the production of nutraceuticals. However, a wide range of applications of lactobacilli in food biotechnology remains potential, whereas a number of important strains still need to be discovered and characterized. This article provides an overview of the taxonomy of lactobacilli and describes four of the most significant case studies on the application of this group of LAB in food and feed biotechnology, including their use as probiotics, dairy starters, silage inoculants, and microbial cell factories. The importance of access to and exchange of biological material within and between different strain collections as a crucial step in expanding the range of different biotechnological applications of lactobacilli is also emphasized. 2010 Elsevier Masson SAS. All rights reserved.

Keywords: Lactobacillus spp.; Probiotic lactobacilli; Dairy starter cultures; Silage inoculants; Microbial cell factories

1. Introduction

The genus Lactobacillus belongs to the large group of lactic acid bacteria (LAB) which are all Gram-positive organisms which produce lactic acid by fermentation. Genera of LAB include, among others, Lactococcus , Enterococcus , Oeno- coccus , Pediococcus , Streptococcus , Leuconostoc and Lacto- bacillus ( Kandler and Weiss, 1986 ). With over 100 species and subspecies, the genus Lactobacillus represents the largest group within the family Lactobacillaceae. Members of the genus are rod-shaped, often organized in chains. They are strictly fermentative and aerotolerant, but grow well under anaerobic conditions. There are two groups of species depending on the ability to ferment sugars: homofermentative species, converting sugars mostly into lactic acid, and heterofermentative species,

* Corresponding author. E-mail addresses: giorgio.giraffa@entecra.it (G. Giraffa), n_chanish. ibmv@caucasus.net (N. Chanishvili), yantyatiwidyastuti@yahoo.com (Y. Widyastuti).

0923-2508/$ - see front matter 2010 Elsevier Masson SAS. All rights reserved.

doi:10.1016/j.resmic.2010.03.001

converting sugars into lactic acid, acetic acid, ethanol and CO 2 . Because the main catabolite is lactic acid, lactobacilli prefer relatively acidic conditions (pH 5.5 e 6.5). Bacteria belonging to the genus Lactobacillus (lactobacilli) can be found in a variety of ecological niches such as plants, animals and raw milk ( Hammes and Vogel, 1995 ). In addition, lactobacilli can be found in insects. The ability to colonize such a variety of habitats is a direct consequence of the wide metabolic versatility of this group of LAB. Hence, it is not unexpected that lactobacilli have been used for decades in food preservation, as starters for dairy products, fermented vegetables, fish and sausages as well as silage inoculants. Because of their potential therapeutic and prophylactic attri- butes, lactobacilli have also been proposed as probiotics.

1.1. Taxonomy and phylogeny

In addition to their ecological versatility, the diversity of Lactobacillus is reflected in the considerable phenotypic and genotypic variation within the genus. Comparative analysis of

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16S/23S rRNA gene sequences revealed phylogenetic rela- tionships among lactobacilli. Collins et al. (1991) originally distinguished three phylogenetic groups, the Lactobacillus casei e Pediococcus , Lactobacillus delbrueckii, and Leuco- nostoc group. Later, the L. delbrueckii group was renamed as the Lactobacillus acidophilus group ( Schleifer and Ludwig, 1995 ) and the L. casei e Pediococcus group was split into the L. casei group, the Lactobacillus plantarum group, the Lactobacillus reuteri group, the Lactobacillus buchneri group and the Lactobacillus salivarius group. At present, the phylogenetic structure of the genus Lactobacillus also includes the Lactobacillus perolens group, the Pediococcus group, and the Lactobacillus vitulinus e catenaformis group ( Pot and Tsakalidou, 2009 ). The L. acidophilus group contains almost exclusively obligately homofermentative lactobacilli. Besides L. acid- ophilus, many other species of industrial interest such as Lactobacillus amylovorus , Lactobacillus crispatus , L. del- brueckii with the subspecies delbrueckii , lactis , bulgaricus , and indicus, Lactobacillus johnsonii , Lactobacillus helveticus , and Lactobacillus gasseri are included. L. helveticus and L. delbrueckii play an acknowledged role as starters in dairy and vegetable fermentation, respectively, whereas some strains of the species L. acidophilus have long been known to play a key role in human health and nutrition by their positive influence on the intestinal flora ( Pot, 2008 ). The L. casei group contains the most well-known species Lactobacillus rhamnosus, Lactobacillus paracasei, L. casei, and Lactobacillus zeae . This subgroup has been subjected to exten- sive taxonomic revision in the past, leading to a provisional rejection of the species name L. paracasei. At the moment, these species are still considered separate species within the genus (Dellaglio et al., 2002; Desai et al., 2006; De Vos et al., 2005; Pot, 2008 ). L. paracasei and L. rhamnosus are the most common foodborne isolates within the L. casei group of lacto- bacilli, especially from cheese. L. paracasei and L. casei are also found in silage and are common inhabitants of the animal/human gastrointestinal tract (Pot and Tsakalidou, 2009 ). The L. plantarum group encompasses the taxa L. plantarum subsp. plantarum , L. plantarum subsp. argentoratensis , Lacto- bacillus paraplantarum , and Lactobacillus pentosus . L. plan- tarum is widely found in a range of food such as dairy, meat, and vegetable products. It is commonly found in the human gastrointestinal tract as a consequence of its proven ability to survive gastric transit and colonize the gut. L. plantarum is considered a food-grade microorganism because of its long and documented history of safe use in fermented foods ( de Vries et al., 2006 ). The L. reuteri group contains at least six species exclusively isolated from sourdough. It should be noted that L. reuteri strains may also be dominant members of sourdough pop- ulations (Del Bello et al., 2005 ). Lactobacillus fermentum , one of the best-known species of this group, has been isolated from vegetable and dairy fermentation ( Pot, 2008; Pot and Tsakalidou, 2009 ). The L. buchneri group is a large and heterogeneous group of lactobacilli mostly linked to food fermentations. To this

group belong species isolated from vegetable fermentation ( L. buchneri , Lactobacillus brevis , Lactobacillus hilgardii ), sourdough ( Lactobacillus sanfranciscensis ) and kefir grains ( Lactobacillus kefiri and Lactobacillus parakefiri ). The L. salivarius group is a very heterogeneous group. L. salivarius is a homofermentative Lactobacillus isolated from the oral cavity of man. Lactobacillus algidus was isolated as part of the predominant psychrophilic flora from vacuum- packaged beef. Lactobacillus agilis was shown to be the main component of the pigeon crop flora ( Pot, 2008; Pot and Tsakalidou, 2009 ).

2. Application of lactobacilli in food and feed biotechnology

Lactobacilli are associated with food production because of the preservative action due to acidification, and/or enhance- ment of flavor, texture and nutrition. Lactobacilli are used as starters or complementary cultures for several varieties of cheese, fermented plant foods, fermented meats, in wine and beer production, sourdough bread and silage. They cause rapid pH decrease in the raw material through the production of lactic acid as the main catabolic product. In addition, the proteolytic activity and production of aroma compounds, bacteriocins and exopolysaccharides are relevant for the quality and nutritional value of the end product and expand the spectrum of biotechnological applications of this important group of LAB ( Leroy and De Vuyst, 2004 ). Some lactobacilli of the gastrointestinal tract (GIT) have also been associated

with health benefits, which have given rise to their designation

as probiotics. At present, the consumer is paying considerable attention to the relationship between food and health. As

a consequence, the demand for functional foods, i.e. foods

claimed to possess health-promoting properties beyond basic nutrition, has dramatically increased over the last years ( Keohane et al., 2009; O’May and MacFarlane, 2005; Ouwehand et al., 2002 ). Bacteriocin and exopolysaccharide production are two meaningful examples of functional applications of lactobacilli in the food industry. In the last two decades, several studies have demonstrated the potential of bacteriocins to control growth of pathogenic microorganisms in food products. Bacteriocin- producing lactobacilli have been suggested as protective cultures in fermented meats, fermented olives, and dairy prod- ucts (Hammes and Vogel, 1995; Leroy and De Vuyst, 2004). Plant or microbial polysaccharides are widely used in the food industry. They are known to increase viscosity and firmness, improve the texture and contribute to the taste of low-fat prod- ucts. The in situ production of exopolysaccharides has been reported in several lactobacilli (Leroy and De Vuyst, 2004 ). These few examples suggest that lactobacilli are poly- functional bacteria with industrially important implications. The following paragraphs will provide more detailed insight through four case studies on the most significant applications of lactobacilli in food and food biotechnology: lactobacilli as probiotics; lactobacilli as dairy starters; lactobacilli as silage inoculants; and lactobacilli as microbial cell factories.

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2.1. Lactobacilli as probiotics

2.1.1. Definition and mechanism of action of probiotics

Probiotics are defined by FAO/WHO (2002) as ‘‘live microorganisms which, when administered in adequate amounts, confer health benefit on the host’’. There is solid scientific evidence to support th e concept that the maintenance of healthy gut microflora may provide protection against gastrointestinal disorders, in cluding gastrointestinal infec- tions and inflammatory bowel d iseases. Many studies have also demonstrated the efficiency of probiotics at offering an appropriate alternative to the use of antibiotics in the treat- ment of enteric infection ( Marteau et al., 2001 ) or to reduce the symptoms of antibiotic-associated diarrhea ( Rastall et al., 2005 ). Probiotic bacterial cultures modulate the growth of intestinal microbiota, suppress potentially harm ful bacteria and reinforce the body’s natural defense mechanisms. Currently, much evidence exists on the positive effects of probiotics on human health ( Mattila-Sandholm et al., 2002; Millette et al., 2008; O’May and MacFarlane, 2005; Parvez et al., 2006 ).

2.1.2. Selection and application

Lactobacilli are the most extensively studied and widely used probiotics within the LAB. Most Lactobacillus strains belong to the L. acidophilus group. L. (para)casei , L. plantarum , L. reuteri , and L. salivarius , which represent the respective phylogenetic groups, are known to contain probiotic strains. In order for a probiotic to be of benefit to human health, it must fulfill several criteria. It must survive passage through the upper GIT and reach its site of action alive, and it must be able to function in the gut environment. The functional require- ments of probiotics include tolerance to human gastric juice and bile, adherence to epithelia l surfaces, persistence in the human GIT, immune stimulation, antagonistic activity toward intestinal pathogens (such as Helicobacter pylori , Salmonella spp., Listeria monocytogenes , and Clostridium difficile ), and the capacity to stabilize and modu late the intestinal microbiota ( Mattila-Sandholm et al., 2002; Millette et al., 2008; O’May and MacFarlane, 2005 ). Several technological aspects have to be considered in probiotic selection. These include good sensory properties, phage res istance, viability during pro- cessing and stability in production and du ring storage. Pro- biotics must be non-pathogenic and non-toxic and should not carry acquired, genetically exchangeable antibiotic resistance ( Mattila-Sandholm et al., 2002; Ouwehand et al., 2002 ).

2.1.3. Overview of probiotic food products containing

lactobacilli Dairy products are, however, the most widely used food carriers for delivering probiotics. The probiotic microorganisms most often belong to L. acidophilus, L. gasseri, L. helveticus , L. johnsonii , L. (para)casei , L. reuteri, L. plantarum , L. rham- nosus , and L. fermentum (Table 1 ) (for a review, see Tamime et al., 2005 ). A wide range of probiotic dairy products is available in different markets; typical examples include pasteurized milk, ice cream, fermented milks, cheeses, and

baby feed milk powder. Yoghurt is the classical example of probiotic fermented milk. The manufacturing stages of pro- biotic yoghurt are very similar to ‘‘classical’’ yoghurt, but the fermentation time is slightly longer compared with the ‘‘clas- sical’’ product ( Tamime et al., 2005 ). Due to its limited acidity, low oxygen level, high lipid content and low storage temperature, cheese also appears to be a suitable carrier for delivering live probiotic bacteria (Grattepanche et al., 2008 ). Probiotic L. paracasei and L. rhamnosus GG have been used in cheddar and cottage cheese manufacturing. Usually, probiotic bacteria are introduced into cheese as adjunct cultures along with the lactic starter cultures (Tamime et al., 2005 ).

2.1.4. Research prospects and future applications Although it has been extensively demonstrated that dairy fermented products are the best matrices for delivering pro- biotics, there is growing evidence of the possibility of obtaining probiotic foods from non-dairy matrices. Several raw materials (such as cereals, fruits, and vegetables) have recently been investigated to determine their suitability for designing new, non-dairy probiotic foods (for a review, see Rivera-Espinoza and Gallardo-Navarro, 2010). Most presently available probiotics belong to the genus Lactobacillus . However, few strains are commercially avail- able for probiotic application (Table 1 ). Gene technology and comparative genomics will play a role in rapid searching and developing of new strains, with gene sequencing allowing for an increased understanding of mechanisms and the function- ality of probiotics (Reid, 2008 ).

Table 1 The most commonly used species of lactobacilli in probiotic preparations (especially in dairy products) (Keohane et al., 2009; Ouwehand et al., 2002; Parvez et al., 2006; Rastall et al., 2005).

Species

Main commercially used strain(s) (when available) with documented health benefits

Lactobacillus acidophilus Lactobacillus johnsonii Lactobacillus (para)casei Lactobacillus rhamnosus

LA1; LA5 La1; NCFM; DDS-1; SBT-2062 F19; CRL 431; Immunitass; Shirota GG; LB21; 271; GR-1; VTT E-97800 299v; Lp01

Lb12

Lactobacillus plantarum Lactobacillus delbrueckii subsp. bulgaricus Lactobacillus delbrueckii subsp. lactis Lactobacillus cellobiosus Lactobacillus curvatus Lactobacillus fermentum Lactobacillus reuteri Lactobacillus brevis Lactobacillus salivarius Lactobacillus helveticus Lactobacillus amylovorus Lactobacillus crispatus Lactobacillus gallinarum Lactobacillus gasseri

Lla

RC-14

MM2

UCC118

B02

LG21

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2.2. Lactobacilli as dairy starters

2.2.1. Role and use

Starter cultures are microorganisms that are intentionally added to raw material to create a desired outcome in the final product, most often through their metabolic activities. The most common use of starter cultures is for production of lactic acid from lactose (milk sugar), which in most cases causes or assists in the coagulation of milk protein by lowering its pH value. Certain starter organisms are added specifically for their ability to produce flavor compounds such as diacetyl. Starter organisms can also influence flavor and texture of cultured and/or aged products through the breakdown of proteins, fats and other milk constituents in addition to the pH effect. A decreased pH of cultured products can be inhibitory to certain spoilage organisms, although inhibition is also associated with other byproducts such as hydrogen peroxide ( Chamba, 2008 ). The starter cultures used today in the dairy industry are composed of selected strains of LAB, which were originally present as part of the contaminating microflora of milk ( Chamba, 2008; Pot, 2008; Pot and Tsakalidou, 2009 ). Modern starter cultures have developed from the practice of retaining small quantities of whey or cream from the successful manufacture of a fermented product on a previous day and using this as the inoculum or starter for the next day’s production. In another type of fermentation, designated as ‘controlled’ or ‘pure culture fermentation’, the microorgan- isms are first purified from the original food product, identi- fied, and maintained in the laboratory. When required for fermentation, selected strains are grown in high volumes and added to the raw material (e.g. milk) in very high numbers. These cultures, when used in controlled fermentation, are referred to as ‘starter cultures’ ( Parente and Cogan, 2004 ). Traditional cultures are of significant scientific and techno- logical interest. Natural whey starters, despite their unpredict- able performance, are still used extensively, for example, in the manufacture of mozzarella cheese using milk obtained from water buffaloes (Bubalus arnee ) in southern Italy ( Parente and Cogan, 2004 ). Natural starters used for preparation of Mat- soni (a yogurt-like product produced in the Caucasian region) are known to contain lactobacilli, streptococci, sometimes lactococci and small amounts of yeasts (Brusetti et al., 2008;

Merabishvili et al., 1999; Uchida et al., 2007 ).

2.2.2. Selection and application

The Lactobacillus genus is one of the most important in development of dairy starters, which play a crucial rolein souring raw milk and in the production of fermented dairy products such as cheeses and fermented milks (including probiotics) (Leroy and De Vuyst, 2004). Lactobacilli are used as starters in the manufacturing of yoghurt and mozzarella cheese. They are also used as starter adjuncts to promote faster ripening of cheddar and similar cheeses, to reduce the incidence of bitterness and as probiotics in yoghurt-type products (Parente and Cogan, 2004 ). L. delbrueckii subsp. bulgaricus is widely used along with Streptococcus thermophilus as a starter in yoghurt

manufacturing. L. casei is used as a probiotic, but it is also found

in some starter cultures and commonly belongs to the non-starter

lactic acid bacteria (NSLAB) found in cheddar cheese. L. hel- veticus is frequently used along with other thermophilic LAB in the manufacturing of a range of cheeses including emmental, Grana Padano, Parmigiano Reggiano and mozzarella.

2.2.3. Starter performance and activity

Starter activity can be influenced by a number of factors, including the age of the culture, handling and storage prac- tices, incubation temperature, the quality of the raw milk, bacteriophage attack and the presence of inhibitors such as drugs or sanitizers. High product loss, especially in cheese manufacturing practice, is usually associated with bacterio- phages ( Parente and Cogan, 2004 ). Since phages are strain- specific, a culture rotation strategy is necessary to control phage multiplication. Phages can enter the dairy plant through the raw milk supply, although some lysogenic strains them- selves are ‘‘phage carriers’’. Problems with ‘‘dead vats’’ due to phages can often be linked to phages in the plant environment (e.g. poor plant hygiene, residual culture). Stringent culture handling and plant sanitation programs are essential in pre- venting phage problems ( Kutter and Sulakvelidze, 2004 ).

2.3. Lactobacilli as silage inoculants

2.3.1. Silage making

Silage is an important feed for livestock during food short- ages, not only in winter in cold and temperate regions, but also during the dry season in the tropics ( Mannetje, 1999 ). The purpose of silage making is to preserve fresh forages while minimizing loss of nutrients and avoiding adverse changes in the chemical composition of the ensiled forages. It is therefore

a suitable feed for maintaining the productivity of the livestock. Silage is a product of fermentation which is carried out by LAB under anaerobic conditions. In such an environment, LAB convert water-soluble carbohydrates (WSCs) of the forages into organic acids, mainly lactic acid. The production of these acids reduces the pH of the ensiled forages and inhibits growth of aerobic spoilage microorganisms ( McDonald et al., 1991 ). The fermentation of silage requires several weeks for completion, with a critical time normally taking only a few hours (Oude Elferink et al., 1999 ). Silage fermentation is

a complex and sometimes unpredictable process because it

involves many species of LAB where interactions among them may occur ( Table 2). It is commonly recommended to add bacterial inoculants, especially those based on lactobacilli, to ensure the fermentation process. Fig. 1 reports different steps in silage preparation and fermentation, which is usually applied in plastic bags.

2.3.2. The role of inoculants

Silage inoculants are selected lactobacilli added to ensiled forage to dominate or outnumber the naturally epiphytic LAB present in the forage. Both homofermentative and hetero- fermentative lactobacilli have potential advantages as silage inoculants. At the beginning of fermentation, production of

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Table 2 Lactic acid bacteria in silage.

Species

Forage

Reference

Lactobacillus plantarum Lactococcus lactis Leuconostoc pseudomesenteroides Pediococcus acidilactici Lactobacillus brevis Enterococcus faecalis Weissella kimchii Pediococcus pentosaceus Lactobacillus plantarum Pediococcus pentosaceus Lactobacillus homohiochii Lactobacillus brevis Lactobacillus gasseri Lactobacillus plantarum Lactobacillus coryniformis Leuconostoc spp. Enterococcus faecium Enterococcus faecalis Pediococcus acidilactici Pediococcus pentosaceus Pediococcus spp. Lactobacillus brevis Lactobacillus fermentum Leuconostoc mesenteroides

Paddy rice ( Oryza sativa)

Ennahar et al., 2003

Alfalfa ( Medicago sativa L.)

Lin et al., 1992

Pangola ( Digitaria decumbens), Setaria ( Setaria sphacelata) and Hamil ( Panicum maximum)

Tjandraatmadja et al., 1994a,b

lactic acid by homofermentative lactobacilli is preferred to reduce pH faster, which may inhibit growth of undesirable microorganisms and improve fermentation quality ( Cai et al., 1999 ). Good aerobic stability is then controlled by the heter- ofermentative lactobacilli, since the activity of yeast is impaired due to acetic acid produced ( Driehuis et al., 2001; Filya, 2003 ). Combining homofermentative and hetero- fermentative inoculants has become popular and has been used for various forages ( Filya, 2003; Weinberg et al., 1999; Zhang et al., 2009 ).

2.3.3. Selection and application There are many commercial inoculants available on the market. They may vary and be based on several requirements such as their ability to dominate the natural population of microorganisms in the forages and to produce lactic acid

rapidly as a result of effective fermentation, which leads to

a drastic drop in pH. It is also essential that inoculants be

stable during storage so that the correct bacterial concentration

is applied to the ensiled forage. Several strains of L. plantarum

(homofermentative) and L. buchneri (heterofermentative) have been selected and developed as silage inoculants. Ideally, the inoculants should provide 10 6 CFU per g of fresh crop forages (McDonald et al., 1991 ). This concentration

crop forages ( McDonald et al., 1991 ). This concentration Fig. 1. Silage preparation using lactobacilli

Fig. 1. Silage preparation using lactobacilli as inoculants. A: chopping grass; B: adding rice bran and inoculants as additives; C: silage inoculants; D: silage fermentation in plastic bags.

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may decrease, since their viability declines with increasing

temperature. Higher temperature results in even greater losses

in viability for some inoculants. Mulrooney and Kung (2008)

reported that L. plantarum MTD/1 was the most thermoto-

lerant inoculant. Selection of thermotolerant silage inoculants

is necessary for the best performance of this practice in the

tropics. L. plantarum CA28 grew well during the ensilage of perennial ryegrass at 25 C (Zhang et al., 2000 ).

2.3.4. Future prospects

Although silage making has been practiced since ancient times, research to improve the quality of inoculants for silage

has been intensively carried out. The aim is to find strains with improved properties such as antibacterial activity and pro- biotic potential. Among available silage inoculants, L. plan- tarum MTD1 (Ecosyl), and L. plantarum (Agri-King) showed strong antibacterial activity against Micrococcus luteus , while L. buchneri (both of Biotal and Pioneer Hi-Bred International) was effective against Pseudomonas aeruginosa ( Gollop et al., 2005 ). Several studies of Weinberg et al. (1999, 2003, 2004a,b) showed that some lactobacilli as silage inoculants were able to survive in the rumen fluid. Their survival is

a good indication that they might have probiotic effects upon the host animals. The development of genetically modified L. plantarum as

a silage inoculant expressing cellulases and xylanases has

proven to be attractive as an alternative to the direct addition

of hydrolytic enzymes during ensiling. Cellulolytic recombi-

nant L. plantarum modified by integration of cel A gene

encoding an alkaline endo-1,4-b glucanase from Bacillus sp.

in their chromosome showed improved acidification ability for

alfalfa (Rossi et al., 2001 ).

2.4. Lactobacilli as microbial cell factories

Through their metabolic activity, lactobacilli may act as

‘‘cell factories’’ for the de novo generation of bioactivities from

a range of food protein sources. Biological activities associated with such peptides include immunomodulating, antibacterial, antihypertensive and opioid-like properties. Milk proteins are

recognized as a primary source of bioactive peptides, which can

be encrypted within the amino acid sequence of dairy proteins,

requiring proteolysis for release and activation. These obser- vations have highlighted interest in developing value-added fermented foods that are selectively enriched with compounds known to deliver a particular health benefit to the consumer, such as the antihypertensive angiotensin-converting enzyme (ACE) inhibitors, which are produced by lactobacilli through their proteolytic system ( Broadbent, 2008; Hayes et al., 2007 ).

2.4.1. Metabolic engineering

Metabolic engineering may be an alternative tool when searching for new strains with improved biotechnological characteristics, e.g. for the production of nutraceuticals. Meta- bolic engineering strategies of LAB may lead to the efficient rerouting of sugar metabolism to nutritional end-products other than lactic acid, such as aroma compounds, low-calories sugars

and natural sweeteners (Hugenholtz et al., 2002). Different Lactobacillus species have been metabolically engineered for production of L(þ ) lactic acid, mannitol, pyruvate and L-ribulose (Aarnikunnas et al., 2003; Helanto et al., 2007; Nikkila¨ et al.,

2000).

2.4.2. Lactobacilli as live vaccine carriers Similarly to other LAB, lactobacilli can also be efficient oral vaccine carriers. Most of our current knowledge of the use of lactobacilli for vaccination purposes has been obtained with tetanus toxin fragment C as the model antigen. This knowledge, together with our increasing understanding of the immune system and recent advancements in cloning and expression techniques, will make development of lactobacilli as delivery systems for live vaccines a realistic perspective (Seegers, 2002 ).

3. Concluding remarks

The genus Lactobacillus is a heterogeneous group of LAB with important implications in food and feed fermentation. Lactobacilli are currently used as probiotics, silage inoculants and as starters in fermented food. However, a wide range of applications for lactobacilli in food biotechnology is possible, and a number of important strains need to be discovered and characterized. In this respect, access and exchange of biological material within and between different strain collections will be crucial for expanding the range of different biotechnological applications of lactobacilli. This would enable us to dispose of new isolates from different countries and geographic areas which hopefully would carry new and interesting metabolic traits. With reference to the case studies covered here, the availability of new strains could help, for example, to design new probiotic products or starter cultures for the food industry or as inoculants in silage. Undoubtedly, common sharing of genomic information is helping to develop biotechnological-related applications of lactobacilli. The present availability of 25 LAB genome sequences (including 15 Lactobacillus spp.) from public data- bases is providing an expanded view of genetic and metabolic capacities of these bacteria and enabling researchers to perform functional and comparative genomics studies. Genome sequencing and functional genomics studies of a variety of LAB, including lactobacilli, are now rapidly providing insight into their diversity and evolution and revealing the molecular basis for important traits such as flavor formation, sugar metabolism, stress response, adaptation and interactions (Siezen et al., 2004). For example, bioinformatics tools can be used, in a genome- mining approach, to search genomes for essential components such as proteinases, peptidases, aminotransferases, enzymes for amino acid biosynthesis and transport systems for peptides and amino acids which may be involved in flavor-forming reactions in LAB (van Kranenburg et al., 2002). Expanding this approach to other genomes, combined with experimental verification of the predicted substrate specificities, will lead to the design of probes for high-throughput screening and Lactobacillus spp. strain selection in the future.

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References

Aarnikunnas, J., von Weymarn, N., Ro¨ nnholm, K., Leisola, M., Palva, A., 2003. Metabolic engineering of Lactobacillus fermentum for production of mannitol and pure L-lactic acid or pyruvate. Biotechnol. Bioeng. 82, 653e663. Broadbent, J.R., 2008. Proteolysis in lactobacilli: applications in food and health. In: Proceedings of the Ninth Symposium on Lactic Acid Bacteria, August 31eSeptember 4, Egmond aan Zee, The Netherlands. Brusetti, L., Malkhaz ova, I., Mora, D., Borin, S., Merabishvili, M., Zaccaria, A., Colnago, D., Chanishvili, N., Daffonchio, D., 2008. Fluo- rescent-Box-PCR, an improved tool for resolving bacterial genetic diversity and biogeography studies. BMC Microbiol. 8, 220 e 232. Cai, Y., Benno, Y., Ogawa, M., Kumai, S., 1999. Effect of applying lactic acid bacteria isolated from forage crops on fermentation characteristics and aerobic deterioration of silage. J. Dairy Sci. 82, 520 e526. Chamba, J.F., 2008. Application des bacte´ ries lactiques lors des fabrication fromage` res. In: Corrieu, G., Luquet, F.M. (Eds.), Bacte´ ries lactiques e De la ge´ ne´ tique aux ferments. Lavoisier, Paris, pp. 787e815. Collins, M.D., Rodrigues, U.M., Ash, C., Aguirre, M., Farrow, J.A.E., Mar- tinez-Murcia, A., Phillips, B.A., Williams, A.M., Wallbanks, S., 1991. Phylogenetic analysis of the genus Lactobacillus and related lactic acid bacteria as determined by reverse transcriptase sequencing of 16S rRNA. FEMS Microbiol. Lett. 77, 5e12. Del Bello, F., Walter, J., Roos, S., Jonsson, H., Christian, H., 2005. Inducible gene expression in Lactobacillus reuteri LTH5531 during type I.I. sour- dough fermentation. Appl. Environ. Microbiol. 71, 5873e5878. Dellaglio, F., Felis, G.E., Torriani, S., 2002. The status of the species Lactobacillus casei (Orla-Jensen 1916) Hansen and Lessel 1971 and Lactobacillus paracasei Collins et al. 1989: request for an opinion. Int. J. Syst. Bacteriol. 52, 285e287. Desai, A.R., Shah, N.P., Powell, I.B., 2006. Discrimination of dairy industry isolates of the Lactobacillus casei group. J. Dairy Sci. 89, 3345 e3351. De Vos, P., Tru¨ per, H.G., Tindall, B.J., 2005. Judicial commission of the international committee on systematics of prokaryotes, 10th international congress of bacteriology and applied microbiology, minutes of meetings 28, 29 and 31 July and 1 August 2002, Paris, France. Int. J. Syst. Evol. Microbiol. 55, 525e532. de Vries, M., Vaughan, E., Kleerebezem, M., de Vos, W.M., 2006. Lactoba- cillus plantarum e survival, functional and potential probiotic properties in the human gastrointestinal tract. Int. Dairy J. 16, 1018e 1028. Driehuis, F., Oude Elferink, S.J.W.H., VanWikselaar, P.G., 2001. Fermentation characteristics and aerobic stability of grass silage inoculated with Lactobacillus buchneri, with or without homofermentative lactic acid bacteria. Grass Forage Sci. 56, 330e343. Ennahar, S., Cai, Y., Fujita, Y., 2003. Phylogenetic diversity of lactic acid bacteria associated with paddy rice silage as determined by 16S ribosomal DNA analysis. Appl. Environ. Microbiol. 69, 444e451. FAO/WHO, 2002. Guidelines for the Evaluation of Probiotics in Food. Working group report. Food and Agricultural Organization of the United Nations and World Health Organization, London, Ontario, Canada. Filya, I., 2003. Effect of Lactobacillus buchneri and Lactobacillus plantarum on the fermentation, aerobic stability and ruminal degradability of low dry matter corn and sorghum silages. J. Dairy Sci. 86, 3575 e3581. Gollop, N., Zakin, V., Weinberg, Z.G., 2005. Antibacterial activity of lactic acid bacteria included in inoculants for silage and in silages treated with these inoculants. J. Appl. Microbiol. 98, 662 e666. Grattepanche, F., Miescher-Schwenninger, S., Meile, L., Lacroix, C., 2008. Recent developments in cheese cultures with protective and probiotic functionalities. Dairy Sci. Technol. 88, 421e444. Hammes, W.P., Vogel, R.F., 1995. The genus Lactobacillus. In: Wood, B.J.B., Holzapfel, W.H. (Eds.), The Genera of Lactic Acid Bacteria, vol. 2. Blackie Academic and Professional, London, pp. 19e54. Hayes, M., Ross, R.P., Fitzgerald, G.F., Stanton, C., 2007. Putting microbes to work: dairy fermentation, cell factories and bioactive peptides. Part I:

overview. Biotechnol. J. 2, 426 e434. Helanto, M., Kiviharju, K., Leisola, M., Nyysso¨ la¨ , A., 2007. Metabolic engineering of Lactobacillus plantarum for production of L-ribulose. Appl. Environ. Microbiol. 73, 7083e 7091.

Hugenholtz, J., Sybesma, W., Groot, M.N., Wisselink, W., Ladero, V., Burgess, K., van Sinderen, D., Piard, J.C., Eggink, G., Smid, E.J., Savoy, G., Sesma, F., Jansen, T., Hols, P., Kleerebezem, M., 2002. Meta- bolic engineering of lactic acid bacteria for the production of nutraceut- icals. Antonie van Leeuwenhoek 82, 217e235. Kandler, O., Weiss, N., 1986. Genus Lactobacillus Beijerinck 1901, 212A.L. In: Sneath, P.H.A., Mair, N.S., Sharpe, N.E., Holt, J.H. (Eds.), Bergey’s Manual of Systematic Bacteriology, vol. 2. Williams and Wilkins, Balti- more, pp. 1209e1234. Keohane, J., Ryan, K., Shanahan, F., 2009. Lactobacillus in the gastrointestinal tract. In: Ljungh, A., Wadstro¨ m, T. (Eds.), Lactobacillus Molecular Biology:

From Genomics to Probiotics. Caister Academic Press, Norfolk, pp. 169e181. Kutter, E., Sulakvelidze, A., 2004. Bacteriophages: Biology and Applications. CRC Press, London. Leroy, F., De Vuyst, L., 2004. Lactic acid bacteria as functional starter cultures for the food fermentation industry. Trends Food Sci. Technol. 15, 67 e78. Lin, C., Bolsen, K.K., Brent, B.E., Fung, D.Y.C., 1992. Epiphytic lactic acid bacteria succession during pre-ensiling and ensiling periods of alfalfa and maize. J. Appl. Bacteriol. 73, 375e387. Mannetje, L.’t, 1999. Introduction to the conference on silage making in the tropics. In: Mannetje, L.’t (Ed.), Silage Making in the Tropics with Particular Emphasis on Smallholders. Proceedings of the FAO Electronic Conference on Tropical Silage, 1 Septembere15 December, Rome, Italy. Marteau, P.R., De Vrese, M., Cellier, C.J., Schrezenmeir, J., 2001. Protection from gastrointestinal diseases with the use of probiotics. Am. J. Clin. Nutr. 73, 430e436. Mattila-Sandholm, T., Mylla¨ rinen, P., Crittenden, R., Mogensen, G., Fonde´ n, R., Saarela, M., 2002. Technological challenges for future pro- biotic foods. Int. Dairy J. 12, 173 e182. McDonald, P., Henderson, N., Heron, S., 1991. The Biochemistry of Silage, second ed. Chalcombe Publications, Southampton. Merabishvili, M., Prochkhidze, K., Natroshvili, G., Chanishvili N., 1999. Traditional Caucasian yogurt-like food product Matsoni e chemical and microbial characteristics. In: Proceedings of the 17th International Conference of the International Committee on Food Microbiology and Hygiene, September 13e17, Veldhoven, the Netherlands. Millette, M., Luquet, F.M., Ruiz, M.T., Lacroix, M., 2008. Characterization of probiotic properties of Lactobacillus strains. Dairy Sci. Technol. 88, 695e705. Mulrooney, C.N., Kung Jr., L., 2008. The effect of water temperature on the viability of silage inoculants. J. Dairy Sci. 91, 236e240. Nikkila¨ , K.K., Hujanen, M., Leisola, M., Palva, A.A., 2000. Metabolic engi- neering of Lactobacillus helveticus CNRZ32 for production of pure L- ( þ)-lactic acid. Appl. Environ. Microbiol. 66, 3835e3841. O’May, G.A., MacFarlane, G.T., 2005. Health claims associated with pro- biotics. In: Tamime, A.Y. (Ed.), Probiotic Dairy Products. Blackwell Publishing Ltd, Oxford, pp. 138e166. Oude Elferink, S.J.W.H., Dreiehuis, F., Gottschal, J.C., Spoelstra, S.F., 1999. Silage fermentation processes and their manipulation. In: Mannetje, L.’t (Ed.), Silage Making in the Tropics with Particular Emphasis on Small- holders. Proceedings of the FAO Electronic Conference on Tropical Silage, 1 Septembere15 December, Rome, Italy. Ouwehand, A.C., Salminen, S., Isolauri, E., 2002. Probiotics: an overview on beneficial effects. Antonie van Leeuwenhoek 82, 279e 289. Parente, E., Cogan, T.M., 2004. Starter cultures: general aspects. In: Fox, P.F., McSweeney, P.L.H., Cogan, T.M., Guinee, T.P. (Eds.), Cheese: Chemistry, Physics and Microbiology, vol. I. Chapman and Hall, London, pp. 123 e148. Parvez, S., Malik, K.A., Ah Kang, S., Kim, H.Y., 2006. Probiotics and their fermented food products are beneficial for health. J. Appl. Microbiol. 100,

1171e1185.

Pot, B., 2008. The taxonomy of lactic acid bacteria. In: Corrieu, G., Luquet, F.M. (Eds.), Bacte´ ries lactiques e De la ge´ ne´ tique aux ferments. Lavoisier, Paris. Pot, B., Tsakalidou, E., 2009. Taxonomy and metabolism of Lactobacillus . In:

Ljungh, A., Wadstro¨ m, T. (Eds.), Lactobacillus Molecular Biology: From Genomics to Probiotics. Caister Academic Press, Norfolk, pp. 3e58. Rastall, R.A., Gibson, G.R., Gill, H.S., Guarner, F., Klaenhammer, T.R., Pot, B., Reid, G., Rowland, I.R., Sanders, M.E., 2005. Modulation of the microbial ecology of the human colon by probiotics, prebiotics and

G. Giraffa et al. / Research in Microbiology 161 (2010) 480e487

487

synbiotics to enhance human health: an overview of enabling science and potential applications. FEMS Microbiol. Ecol. 52, 145e152. Reid, G., 2008. Probiotics and prebiotics e progress and challenges. Int. Dairy J. 18, 969e975. Rivera-Espinoza, Y., Gallardo-Navarro, Y., 2010. Non-dairy probiotic prod- ucts. Food Microbiol. 27, 1e 11.

Rossi, F., Rudella, A., Marzotto, M., Dellaglio, F., 2001. Vector-free cloning of

a bacterial endo-1,4-b-glucanase in Lactobacillus plantarum and its effect on

the acidifying activity in silage: use of recombinant cellulolytic Lactobacillus

plantarum as silage inoculant. Antonie van Leeuwenhoek 80, 139e147. Schleifer, K.H., Ludwig, W., 1995. Phylogeny of the genus Lactobacillus and related genera. Syst. Appl. Microbiol. 14, 461e 467. Seegers, J.F.M.L., 2002. Lactobacilli as live vaccine delivery vectors: progress and prospects. Trends Biotechnol. 12, 508e515. Siezen, R.J., van Enckevort, F.H.J., Kleerebezem, M., Teusink, B., 2004. Genome data mining of lactic acid bacteria: the impact of bioinformatics. Curr. Opin. Biotechnol. 15, 105 e115. Tamime, A.Y., Saarela, M., Korslund So¨ ndergaard, A., Mistry, V.V., Shah, N.P.

, 2005. Production and maintenance of viability of probiotic micro-

organisms in dairy products. In: Tamime, A.Y. (Ed.), Probiotic Dairy Products. Blackwell Publishing Ltd, Oxford, pp. 39e 72. Tjandraatmadja, M., Norton, B.W., MacRae, I.C., 1994a. Ensilage of tropical grasses mixed with legumes and molasses.World J. Microbiol. Biotechnol. 10,

82e87.

Tjandraatmadja, M., Norton, B.W., MacRae, I.C., 1994b. Ensilage character- istics of three tropical grasses as influenced by stage of growth and addition of molasses. World J. Microbiol. Biotechnol. 10, 74e81.

Uchida, K., Urashima, T., Chanishvilli, N., Arai, I., Motoshima, H., 2007. Major microbiota of lactic acid bacteria from Matsoni, a traditional fer- mented milk in Georgia. Anim. Sci. J. 78, 85e91. van Kranenburg, R., Kleerebezem, M., van Hylckama Vlieg, J., Ursing, B.M., Smit, B.A., Ayad, E., Smit, G., Siezen, R.J., 2002. Flavour formation from amino acids by lactic acid bacteria: predictions from genome sequence analysis. Int. Dairy J. 12, 111e121. Weinberg, Z.G., Szakacs, G., Ashbell, G., Hen, Y., 1999. The effect of Lactobacillus buchneri and L. plantarum at ensiling, on the ensiling fermentation and aerobic stability of wheat and sorghum silages. J. Ind. Microbiol. Biotechnol. 23, 218 e222. Weinberg, Z.G., Muck, R.E., Weimer, P.J., 2003. The survival of silage inoculant lactic acid bacteria in rumen fluid. J. Appl. Microbiol. 94,

1066e1071.

Weinberg, Z.G., Chen, Y., Gamburg, M., 2004a. The passage of lactic acid bacteria from silage into rumen fluid, in vitro studies. J. Dairy Sci. 87,

3386e3397.

Weinberg, Z.G., Muck, R.E., Weimer, P.J., Chen, Y., Gamburg, M., 2004b. Lactic acid bacteria used in inoculants for silage as probiotics for rumi- nants. Appl. Biochem. Biotechnol. 118, 1e7. Zhang, J.G., Cai, Y., Kobayashi, R., Kumai, S., 2000. Characteristics of lactic acid bacteria isolated from forage crops and their effects on silage fermentation. J. Sci. Food Agric. 80, 1455e1460. Zhang, T., Li, L., Wang, X.-f., Zeng, Z.-h., Hu, Y.-g., Cui, Z.-j., 2009. Effect of Lactobacillus buchneri and Lactobacillus plantarum on fermentation, aerobic stability, bacteria diversity and ruminal degradability of alfalfa silage. World J. Microbiol. Biotechnol. 25, 965e971.