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Probiotics in the Food Industry

Probiotics take on a key role in the functional food industry

The name “Probiotics” is derived from Latin (pro) and Greek (biotic) roots meaning “for life”. Probiotics are defined as live bacteria with beneficial effects on the health of the host organism. Today, primarily lactic acid bacteria (LAB) and bifidobacteria are used as probiotics, however, certain yeasts and bacilli are also known to have positive effects. In most cases, probiotics are produced directly by fermentation in foods such as yogurt or are supplied through dietary supplements. Although the term “Prebiotics” is also used often in such cases, the two terms should not be confused, since “prebiotics” refers to non-digestible compounds that stimulate the growth and/or activity of probiotics in the gut. Prebiotics are frequently used in functional food and typically consist of oligosaccharides. Prebiotics can be found in milk (galactooligosaccharides, or GOS) and plants with dietary fibers, however, they are often produced by fermentation.

At the beginning of the 20th century, probiotics were thought to have a beneficial effect on the host by improving the intestinal microbial balance. At that time, it was observed that lactic acid bacteria inhibit the growth of proteolytic bacteria due to low pH. Through his studies and observations, the Russian biologist Metchnikoff noted that people from cultures that consumed large amounts of fermented milk, for example, those in Bulgaria and the Russian steppes, generally had a higher lifespan. Therefore, he proposed that lactic-acid bacteria decrease the intestinal pH because of fermentation, thereby suppressing the growth of proteolytic bacteria. Metchnikoff propagated the consumption of sour milk fermented with the Lactobacillus bulgaricus 5 as a means of promoting intestinal balance and overall health.

Diverse studies report the benefits of probiotics, such as inhibitory effects on pathogens, aid in the management or prevention of chronic intestinal inflammatory diseases or atopic diseases and support the immune system. Potential beneficial applications abound, and the research around this topic continues to expand.

Recent studies have shown that daily consumption of probiotic cheese helps to bolster immunity in elderly people. Cheese was confirmed to be an effective carrier for probiotics.1

Bifidobacteria and Its health effects

Yogurt sample cultured on BSM Agar. Bifidobacteria appears as purple-brown colonies.

Figure 1.Bifidobacteria as purple-brown colonies in a yogurt sample cultured on BSM Agar.

Bifidobacteriumis one of the most important probiotic bacteria used in the dairy industry. They are Gram-positive, non-motile, rod-shaped, and often branched anaerobic bacteria. They were first isolated from a breast-fed infant by Henry Tissier from the Pasteur Institute. Bifidobacteria have a positive effect on the immune system and help to control intestinal pH. In addition, bifidobacteria produce bacteriocins and bacteriocin-like inhibitory compounds which inhibit the growth of other bacteria.

In adult intestines, only 3-6% of the fecal flora is composed of Bifidobacteria, while in breast-fed infants Bifidobacteria can constitute up to 90%. With increasing age, the number of Bifidobacteria decreases. It was observed that babies and adults with lower numbers of Bifidobacteria have a higher risk for diarrhea and allergies. For this reason, Bifidobacteria are added as a probiotic supplement to infant formulas, drinks, yogurts, and a range of other products.

Bifidobacteria carbohydrate Metabolism

Bifidobacteria possesses many glycosylases that can degrade various plant- or milk-derived oligosaccharides. Several such enzymes were identified on the Bifidobacterium genome. Diverse glycosyl hydrolase, ABC transporter, and the fos gene cluster that is involved in the processing of health-promoting fructooligosaccharides (prebiotics), called bifidogenic factors, can also be found on the genome.10 Bifidobacteria can utilize a broad range of substrates as energy sources, such as plant polymers, glycoproteins and glycoconjugates, while having specialized proteins for the catabolism of oligosaccharides.

Bifidobacteria has a unique hexose metabolism called the bifid shunt. The key enzyme, fructose-6-phosphate phosphoketolase is not found in any other Gram-positive intestinal bacteria and therefore provides an ideal target for a diagnostic test. It was found that live B. lactis bacteria can directly counteract the harmful effects of coeliac-toxic gliadin and this may prove to be a future potential treatment of coeliac disease.9

Bifidobacteria Growth Medium

Because of the wide use of bifidus, we have developed a Bifidobacteria Selective Media (BSM), available as an agar (88517) or a broth (90273), as a standard for quality control. This medium allows for easy and fast quality control of yogurt made with bifidus and can be used to control the count of bifidus bacteria.

Bifidobacterium grow very well on this medium, while Lactobacillus and Streptococcus strains are inhibited. Bifidobacterium colonies grow within 24-48 hours (occasionally up to three days because of the highly selective conditions). The Bifidobacterium colonies are purple-brown and therefore are easy to differentiate from other organisms.

In a Swiss governmental evaluation study for the enumeration of bifidobacteria in sour milk products, the traditional method was compared to Wilkins-Chalgren Agar with 100 mg/L mupirocine and BSM Agar. The traditional method produced statistically significant differences, while Wilkins-Chalgren Agar and BSM Agar showed similar results without any significant variances. The study concluded, “On the BSM Agar, the bifidobacteria forms purple-brown colonies which made the enumeration easy”.3

Table 1. Bifidobacterium sp. cultural characteristics on BSM Agar.
Table 2. Selective medium and supplements for Bifidobacterium.

Lactobacillus Species

3D rendering of isolated Lactobacillus

Figure 2. 3D rendering of isolated Lactobacillus

Lactobacilliare rod-shaped, Gram-positive, fermentative, facultative anaerobic or microaerophilic organotrophs.Lactobacillibelong to the lactic acid bacteria and comprise a major part of this group. As their name implies, they produce lactic acid and derive energy from the fermentation of lactose, glucose and other sugars to lactate via homofermentative metabolism. About 85-90% of the sugar utilized in the fermentative process is converted to lactic acid. However, there are some heterofermentative lactobacilli that produce alcohol in addition to lactic acid from sugars. This acid-producing mechanism inhibits the growth of other organisms and favors the growth of lactobacilli that thrive in low pH environments. ATP is generated during the process by non-oxidative substrate-level phosphorylation.

Some strains of lactobacilli were shown to produce, like bifidobacteria, a bacteriocin-like substance that can inhibit a broad range of pathogens. Lactobacilli also produce adhesins (proteins), which can recognize specific host components (extracellular matrix) vital for bacterial adhesion and colonization at host surfaces, as well as in bacterial interaction with physiological and immunological processes.8

 Probiotic Lactobacillus species include Lactobacillus rhamnosus, Lactobacillus casei, and Lactobacillus johnsonii. Currently, probiotics are not used in the pharmaceutical industry due to the many open questions that are not addressed yet.7

Medium For Lactobacilli

Since lactobacilli prefer acidic conditions, natural extracts and juices from tomatoes and oranges, as well as other single metabolic acids (e.g., malic acid), are often used as media ingredients. Casein and yeast extract provide rich amino acid sources.  Maltose is used as a carbohydrate source for lactobacilli, which cannot utilize glucose as fermentable sugar and fructose is the source of carbohydrate source for Lactobacillus fructivorans. Polysorbate, sorbitan mono-oleate and other related compounds act as a source of fatty acids and stimulate the lactic acid bacteria.

Today, it is standard practice to differentiate lactobacilli based on their phenotype using selective media. Classic phenotypic tests for the identification of lactobacilli are based on physiological characteristics, like motility, growth temperature, respiratory type, and growth in sodium chloride. Other identification factors include diverse biochemical characteristics, such as fermentation type, metabolism of carbohydrate substrates, production of lactic acid isomers, coagulation of milk, and presence of specific enzymes like arginine dihydrolase. In Bergey’s Manual, Lactobacillus is described as a Gram-positive rod, non-spore forming, acid-fast negative and catalase negative. The colony morphology on certain media is taken for the presumptive identification.

Modern Methods for Detection of LACTOBACILLI

As a modern alternative, molecular biology-based methods, like PCR, can be consulted, however, they are often quite expensive. We provide a revolutionary molecular biology method that is rapid, easy, and cost-effective. Based on the detection of rRNA, this method completely avoids the need for PCR amplification. The sandwich hybridization test, called the HybriScan® test, is performed on a microtiter plate. The range of lactobacilli detected by HybriScan® tests is listed in Table 3.

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Table 3.  HybriScan® Products (HybriScan® D = Detection Kit; HybriScan® I = Identification Kit)

For more information visit the technical article page, Lactobacilli Basics, Testing & Identification.


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References

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Ibrahim F, Ruvio S, Granlund L, Salminen S, Viitanen M, Ouwehand AC. 2010. Probiotics and immunosenescence: cheese as a carrier. FEMS Immunol Med Microbiol. 59(1):53-59. https://doi.org/10.1111/j.1574-695x.2010.00658.x
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Rada V. and Koc J. 2000. The use of mupirocin for selective enumeration of bifidobacteria in fermented milk products. Milchwissenschaft. 55,. 65–67.
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Bundesamt für Gesundheit. 2004. Schweizerisches Lebensmittelbuch (SLMB): Kapitel 56, «Mikrobiologie» Neuausgabe 2000, Stand .
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H. Tissier. Recherchers sur la flora intestinale normale et pathologique du nourisson, Thesis, University of Paris (1900).
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G.W. Tannock. 2003. Probiotics: time for a dose of realism, Curr. Issues Intest. Microbiol., 4(2), 33-42.
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Å. Ljungh, T. Wadström. 2009. Lactobacillus Molecular Biology: From Genomics to Probiotics, Caister Academic Press .
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Lindfors K, Blomqvist T, Juuti-Uusitalo K, Stenman S, Venäläinen J, Mäki M, Kaukinen K. Live probiotic Bifidobacterium lactis bacteria inhibit the toxic effects induced by wheat gliadin in epithelial cell culture. 152(3):552-558. https://doi.org/10.1111/j.1365-2249.2008.03635.x
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Kim JF, Jeong H, Yu DS, Choi S, Hur C, Park M, Yoon SH, Kim D, Ji GE, Park H, et al. 2009. Genome Sequence of the Probiotic Bacterium Bifidobacterium animalis subsp. lactis AD011. JB. 191(2):678-679. https://doi.org/10.1128/jb.01515-08
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