Fermented foods: consensus statement and reviewing them

23rd March 2021

Translated from the original article in Catalan

The term “fermented foods” has been widely used but so far has not had a clear definition, there are inconsistencies related to the use of the term “fermented” and is sometimes used including more or less related products, such as probiotics. Although these foods have been consumed for thousands of years, they have recently received increasing attention among biologists, nutritionists, other scientists, and consumers.

In order to develop a definition and describe the role of fermented foods in the human diet, the International Scientific Association for Probiotics and Prebiotics (ISAPP) convened in September 2019 a group of experts to get a consensus on it. ISAPP is a non-profit organization, led by scientists and academics, and although it is funded by companies, its activities are not stipulated by industry. Its mission is to provide objective and scientific information on probiotics, prebiotics and other topics related to nutrition and health.

Figure 1. Website homepage of the International Scientific Association of Probiotics and Prebiotics (ISAPP)

These ISAPP experts have been a total of 13, from the USA, Ireland, Canada, Belgium and South Korea. Most of them are from universities and some companies, and their findings have been published recently (Marco et al 2021).

One of the main conclusions of the consensus they have reached is the definition of fermented foods and beverages: they are those made using the desired microbial growth, which involves enzymatic conversions of food components. In this work, in addition to reviewing what they are, the distinction between fermented and probiotic foods is defined, and the current knowledge about the safety, risks and benefits of these foods is revised. Finally, regulation of fermented foods and the possibility of including them in the dietary guidelines of different countries are reviewed.

What are fermented foods ?

Humans learned a few thousand years ago how to consume and make fermented foods, probably in parallel with the development of agriculture and livestock. See for example my article on fermented cheeses and milks made 7000 years ago.

The consumption of fermented foods spread and promoted from the prehistory to all civilizations especially because it is one of the most effective ways to preserve food, due to the formation of compounds that inhibit other harmful microbes and/or pathogens. These compounds produced by the microorganisms fermenting foods include organic acids (such as lactic or acetic acid), ethanol or bacteriocins. Just remember on the one hand all dairy products that can be stored longer than milk thanks to lactic acid produced by bacteria in cheeses, yogurts, etc. And on the other hand, the consumption of beers or wines as a good hygienic alternative in places and times where uncontaminated running water was not available. The greater sustainability of fermented foods is still very important today in poor regions of the world where there is not enough food security or where there is not access to electricity, refrigeration or clean water.

Other methods of preserving food are decreasing aqueous activity (a_w) by: 1) adding salt or sugar or drying; 2) adding inhibitory compounds (e.g. spices or smoking); 3) vacuuming; and 4) heat treatments (cold or heat), among others.

The other reason for the consumption of fermented foods is the appearance of new organoleptic qualities, such as pleasant and different tastes, smells or textures, due to the biochemical transformations of microorganisms in the composition of the food.

Fermented foods are an important part of the human diet, even in developed regions where food safety and food preservation are well controlled. It has been estimated that more than 5,000 varieties of fermented foods (and beverages) are currently produced and consumed globally (Tamang et al. 2016).

Thus, in the above-mentioned consensus definition of ISAPP, it is very clear that fermented foods are those prepared in a way intended by humans where the activity of microorganisms is required and where they carry out a series of enzymatic reactions of the food components.

Although endogenous or exogenous enzymes, from plants, animals or other sources, may also be present in these foods, this activity is not sufficient to be considered a fermented food, as microbial activity is required.

On the other hand, the main difference with foods spoiled by microorganisms is that these foods are unwanted, and the fermented ones are deliberately made and controlled to generate desirable qualities.

It should also be pointed out that in fermented foods the action of microorganisms is not always through metabolism of fermentation. Indeed, from a biochemical point of view, “fermentation” is a type of metabolism where the energy source and both the electron donor and acceptor are organic compounds (especially carbohydrates), where ATPs are synthesized by phosphorylation of substrate (e.g. in glycolysis) and there is no ATP formation by oxidative phosphorylation with membrane-bound electron transport chains, as would be the case for respiration (Figure 2). Many classic fermented foods are made by fermentative metabolism such as lactic or alcoholic fermentations, but some are also considered fermented foods where microorganisms are doing aerobic respiration, such as acetic vinegar bacteria or molds from some cheeses. Therefore the term “fermented foods” has a broader view, independent of metabolism, where only the active and desired intervention of microorganisms is needed.

Figure 2. Main reactions of the basic mechanisms of ATP synthesis: Oxidative phosphorylation by electron transport chains (top) and Substrate-level phosphorylation (bottom).

Which are the fermented foods and the microorganisms involved ?

We find a good global relationship in the work of Tamang et al (2016), with the microorganisms involved, summarized in Table 1. Of course, this article is not an exhaustive relationship, as for example we miss the traditional Balearic “sobrassada”, although the Sicilian “soppressata” appears in the list, and probably both are related in origin. We see some of the best-known fermented foods in Figure 3.

Table 1. Types of fermented foods and the microorganisms involved (adapted from Tamang et al (2016)

Type	Products	Countries	Microorganisms
Fermented milks and derivatives	Yoghurts, cheeses, buttermilk, kefir, kumis, leben, etc. They are from cows and also from many other mammals, such as sheep, goats, mares, camels, yaks and buffalo	Worldwide	Lactic acid bacteria (LAB) Some bifidobacteria Some yeasts Molds (Penicillium)
Fermented vegetable products (fruits, stems, bulbs, leaves, roots, legumes)	Olives, sauerkraut, kimchi, various pickled vegetables (radishes, aubergines, onions, carrots), fermented cassava, soy products (sauce soy, miso, natto, tempeh). Wines (see below)	Worldwide	LAB Bacillus and other Firmicutes Yeasts Some molds (Rhizopus)
Other fermented vegetable derivatives	Vinegar Fermented tea (i.e. kombucha) and fermented cocoa to make chocolate	Worldwide	Acetic acid bacteria Yeasts
Fermented meat	Sausages such as chorizo, pepper, sausages, sobrassada	Worldwide, especially Europe	LAB Other Firmicutes Some yeasts
Fermented fish and sauces	Very diverse, i.e., nuroc mam, nam plaGarum	East and Southeast Asia Ancient Rome	LAB Other Firmicutes Other bacteria
Alcoholic beverages from cereals, produced with fungal amylolytic cultures	Sake	Japan	Aspergillus oryzae Yeasts
Alcoholic beverages from cereals, produced with human saliva	Chicha	South America	Saliva Yeasts LAB and others
Alcoholic beverages from malt, germinated cereal grain (mainly barley, wheat)	Beer	Worldwide	Yeasts Some lactic acid bacteria
Alcoholic beverages from plant parts	Pulque from agave	Mexico	LAB Zymomonas Yeasts
Alcoholic beverages from fruits	Vine wines	All regions of temperate climate	Yeasts Oenococcus (malolactic fermentation)
Alcoholic beverages from honey	Mead tej	Especially ancient world Ethiopia	Yeasts LAB

As we see in Table 1, the main microorganisms in many fermented foods, from milk to meat and vegetables and others, are lactic acid bacteria (LAB). They are gram-positive bacteria of the phylum Firmicutes (DNA with low G+C), non-spore-forming, aerotolerant anaerobes, and are considered safe. Other bacteria responsible for some fermented foods are Bacillus (sporulated Firmicutes), other Firmicutes such as Staphylococcus, and bacteria of the phylum Actinobacteria (gram-positive with DNA of high G+C) such as Bifidobacterium, Propionibacterium and Brevibacterium. Among the few gram-negative bacteria, the acetic acid bacteria (phylum Alphaproteobacteria) stand out and we must also mention Zymomonas, from the same phylum. You can see the phylogenetic location of all these bacteria in my post “Bacteria: 21 Main phyla, with 147 Important genera”.

In Table 1 we see that besides LAB, the other most important microorganisms in fermented foods are yeasts, especially Saccharomyces, unicellular ascomycete fungus. Other fungi acting in fermented foods are some filamentous ones, such as the ascomycetes Penicillium and Aspergillus, and the zygomycete Rhizopus.

Figure 3. Various fermented foods: sobrassada, cheeses, blue cheese, yogurt, olives, soy sauce, beer and wine.

Living or dead microorganisms in fermented foods ?

Microorganisms that have been actively involved in the processing of fermented foods may be present and viable, that is, alive, in some of these. However, they are absent in other fermented foods because they have been separated from the food or this has had a treatment, usually thermal, removing them (Marco et al 2021).

Among the fermented foods that contain living microorganisms we can mention yogurt, kefir and other fermented milks, most cheeses, miso, natto and tempeh, many of the fermented vegetable products that have not been heat treated such as olives, many of the sausages, kombucha, and some beers.

Fermented foods where microorganisms have been eliminated or removed are for instance bread, heat-treated fermented vegetable products, soy sauce, vinegar, wines, most beers, and coffee and cocoa beans once roasted.

In many uninoculated fermented foods, that is, with their own spontaneous microbiota, there is more than one microorganism responsible for the changes that take place from the original food to the fermented one. There is often a succession of types of microorganisms, depending on the composition of the food and the environmental conditions to which it is subjected: salt, temperature, dryness, etc. For example, in the fermentation of table olives, yeasts and other bacteria first predominate, and finally the LAB end up being imposed.

Differences between fermented foods and probiotics

Although sometimes fermented foods are labelled or named as “probiotics” or “contain probiotics,” it should be made clear that it is not the same in most cases. The term probiotic is only correct to use it when it has been shown to have some beneficial effect on the health of the consumer, and that this effect is due to a living and well-characterized microorganism. This health benefit is beyond the nutritional benefits of the food matrix that contains it. Therefore, the terms “fermented food” and “probiotic” cannot be used by each other.

In the case of fermented foods that may contain some probiotic microorganism, with proven effects on health, it should only be labelled with “contains probiotics” in the event that the probiotic microorganism is well characterized at the strain level and in quantities significant throughout the shelf life of the food.

Fermented foods and food safety

Fermented foods increase the safety of food for the consumer, in the sense that it is more difficult for harmful or pathogenic organisms to grow than with respect to the original foods before fermentation. Indeed, they often contain remarkable amounts of organic acids, such as lactic acid produced by LAB or acetic acid produced by homonymous bacteria. Many of these products at the same time have low water activity, and contain salt and other antimicrobials, making them safe (Adams & Mitchell 2002). Similarly, beverages containing > 4% ethanol or pH < 4.5 are also considered microbiologically safe.

In addition, many LAB, whether native or inoculated, produce bacteriocins that inhibit other undesirable bacteria, such as Listeria or Clostridium.

Some fermented foods also increase safety by removing toxic or antinutrient compounds from raw foods, as is the case with many fermentations of cereals, legumes, and tubers. For example, cassava contains cyanogenic glycosides which are eliminated in fermentation by Lactobacillus plantarum (Lei et al 1999). Also, in the fermentation of the sourdough some LAB facilitate the degradation, by the enzyme phytase (a phosphatase), of phytic acid present in cereals, which is a chelator of divalent cations (Ca, Mg, Fe, Zn) and therefore decreases its adsorption (López et al. 2001).

Furthermore, it can be stated that, with very few exceptions, the microorganisms that are the protagonists of fermented foods, basically LAB, yeasts and filamentous fungi, are not pathogenic nor produce toxic or harmful compounds. In fact, many of them, such as LAB themselves, but also many others (such as some Bacillus, Figure 4) are considered GRAS (generally recognized as safe) by the US FDA or QPS (qualified presumption of safety) by the European EFSA.

Figure 4. One of the last ingredients declared GRAS by the US FDA is precisely a Bacillus subtilis that can also be used in fermented foods. Source: US FDA Gras Notices.

However, as in any type of food, it is always necessary to be very careful, to make sure that the ingredients are fresh and safe, avoid any alteration, and have good controls throughout the production process and in the finished product, checking that there is no contamination of the usual food pathogens.

Some fermented foods contain compounds that can pose food safety risks if consumed in excess. This is the case with alcoholic beverages, which should be taken in moderation due to the effects of ethanol and should be avoided by people at risk. For a different reason and not related to microorganisms, it is also important not to overeat fermented foods that contain salt, such as soy sauce or kimchi.

Some of the few compounds produced by LAB that need to be controlled are biogenic amines, which can be found in small amounts in fermented foods such as cheese, sausages, some vegetables and wine. Biogenic amines can cause various health problems and especially migraines. Their production must be minimized by controlling potential producers species and inoculating non-producing strains.

Mycotoxins such as aflatoxins, ochratoxins and many others, are the main concern of foods fermented with filamentous fungi such as Aspergillus and Penicillium from fermented soybeans, cheeses and others (Sivamaruthi et al 2019). However, in most of these products selected strains are used, either by domestication over centuries or more recently by artificial selection, which do not produce toxins.

Benefits of eating fermented foods on human nutritional health

Beyond food preservation reasons and organoleptic qualities, there is some epidemiological evidence to suggest that diets rich in fermented foods may reduce the risk of disease and increase longevity, health, and quality of life. But these diets, such as the Mediterranean diet, include foods other than fermented ones, and therefore it is not certain that the positive effects are due exclusively to fermented foods. In addition, with the exception of yogurt and other fermented milks, few well-designed and controlled clinical studies have been conducted on the potential health benefits of fermented foods in terms of specific diseases (Marco et al 2021).

However, the indirect health effects of fermented foods are quite apparent when considering the nutritional aspects. Microbial activity leads to the enrichment and / or elimination of various compounds that affect and improve the nutritional composition of the final product.

First, microorganisms reduce the content of high-calorie sugars, id est, monosaccharides and disaccharides, present in milk, meat and vegetables. This reduces the glycemic index and reduces food intolerance, such as lactose in dairy products, wheat fructans, or raffinose and stachyose from legumes. Fermentation hydrolyses polysaccharides, proteins and fats, which facilitates digestion, and as mentioned, it removes various toxic or antinutrient compounds such as phytic acid.

In the case of foods containing polyphenols, lactobacilli have been shown to increase the bioavailability of flavonoids, tannins, and other bioactive compounds. The biosynthesis of vitamins, amino acid derivatives, organic acids such as lactic acid, peptides and cofactors by food-fermenting microbes is well known (Melini et al 2019).

It has been shown that many of the living microorganisms in fermented foods can survive gastric transit and reach the colon, as for example many LAB are tolerant of acidic pH and bile salts and have been shown to be able to maintain transiently in the colon (Elli et al. 2006). Although these microorganisms are unlikely to survive long, it has been shown that they may be metabolically active in the gastrointestinal tract, and that this short-term colonization would be sufficient to produce bioactive compounds, inhibit pathogens, and positively influence the immune system. These effects are increased if there is a daily and repeated consumption of fermented food.

Fermented foods, and the microorganisms they contain, have also been shown to influence the composition of the intestinal microbiota itself (Taylor et al 2020). See also González et al in 2019 and Le Roy et al in 2020. Another additional positive factor in the case of fermented vegetable foods is that many components of these are prebiotics and therefore favour the intestinal microbiota.

In addition, we must take into account the importance of what we eat, including fermented foods, in relation to the immune system. In humans and other mammals 70% of this system is in the gastrointestinal tract, and food is the main source of contact between external antigens and our body. This is particularly important in infants and the initial microbial colonization of the digestive tract. Ingestion of fermented foods during the early years of childhood has been associated with a reduced risk of childhood atopy (genetic predisposition to allergies) (Alm et al. 1999). For any age, it seems that the microorganisms of fermented foods and their components, such as glycopeptide, surface proteins, exopolysaccharides, lipoteichoic acid, or D-phenyl-lactic acid from LAB (Peters et al. 2019) are beneficial for the immune system, especially more demonstrated in fermented milks (Bourrie et al. 2016; Foligne et al. 2016).

In Figure 5 we see a diagram of the basic mechanisms of the possible benefits of fermented foods.

Figure 5. Basic mechanisms of the health benefits of fermented foods, especially from a nutritional point of view, with the transformations of food components into bioactive substances. SCFAs are short-chain fatty acids. Source: Marco et al 2021.

Finally, to conclude, it is necessary to remind that although fermented foods are consumed worldwide and account for approximately 1/3 of the human diet, they are usually absent as recommended foods in diet guidelines (Marco et al 2021). It is also a pity that most of the information that comes out in the media or in popular magazines or on social media about this type of food is exaggerated or wrong, often making them synonymous with probiotics.

Bibliography

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Alm J S, Swartz J, Lilja G, Scheynius A, Pershagen, G (1999) Atopy in children of families with an anthroposophic lifestyle. Lancet 353, 1485–1488

Bourrie B C, Willing B P, Cotter P D (2016) The microbiota and health promoting characteristics of the fermented beverage kefir. Front Microbiol 7, 647

Elli M et al (2006) Survival of yogurt bacteria in the human gut. Appl Environ Microbiol 72, 5113–5117

Foligne B et al (2016) Immunomodulation properties of multi-species fermented milks. Food Microbiol 53, 60–69

González S et al (2019) Fermented dairy foods: impact on intestinal microbiota and health-linked biomarkers. Front Microbiol 10, 1046.

Iraporda C. et al (2015) Lactate and short chain fatty acids produced by microbial fermentation downregulate proinflammatory responses in intestinal epithelial cells and myeloid cells. Immunobiology 220, 1161–1169

ISAPP, The International Scientific Association for Probiotics and Prebiotics: https://isappscience.org

Lei V, Amoa-Awua WK, Brimer L (1999) Degradation of cyanogenic glycosides by Lactobacillus plantarum strains from spontaneous cassava fermentation and other microorganisms. Int. J. Food Microbiol. 53, 169–184

Le Roy C I et al (2020) Red wine consumption associated with increased gut microbiota α-diversity in 3 independent cohorts. Gastroenterology 158, 270–272

López HW et al (2001) Prolonged fermentation of whole wheat sourdough reduces phytate level and increases soluble magnesium. J. Agric. Food Chem. 49, 2657–2662

Marco ML, Sanders ME, Gänzle M et al (2021) The International Scientific Association for Probiotics and Prebiotics (ISAPP) consensus statement on fermented foods. Nature Rev Gastroenterol Hepatol. https://www.nature.com/articles/s41575-020-00390-5

Melini F, Melini V, Luziatelli F, Ficca AG, Ruzzi M (2019) Health-promoting components in fermented foods: an up-to-date systematic review. Nutrients 11, 1189

Peters A et al. (2019) Metabolites of lactic acid bacteria present in fermented foods are highly potent agonists of human hydroxycarboxylic acid receptor 3. PLoS Genet. 15, e1008145

Sivamaruthi BS, Kesika P, Chaiyasut C (2019) Toxins in fermented foods: prevalence and preventions – A mini review. Toxins 11, 4

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Tarvainen M, Fabritius M, Yang B (2019) Determination of vitamin K composition of fermented food. Food Chem 275, 515–522

Taylor B C et al (2020) Consumption of fermented foods is associated with systematic differences in the gut microbiome and metabolome. mSystems 5, e00901-19

Bacillus as probiotics

12th August 2017

The probiotics

Probiotics are living microorganisms that, when ingested in adequate amounts, can have a positive effect on the health of guests (FAO / WHO 2006; World Gastroenterology Organization 2011, Fontana et al., 2013). Guests can be humans but also other animals. Lactic acid bacteria, especially the genus Lactobacillus and Bifidobacterium, both considered as GRAS (Generally recognized as safe), are the microbes most commonly used as probiotics, but other bacteria and some yeasts can also be useful. Apart from being able to be administered as medications, probiotics are commonly consumed for millennia as part of fermented foods, such as yoghurt and other dairy products (see my article “European cheese from 7400 years ago..” “December 26th, 2012). As medications, probiotics are generally sold without prescription, over-the-counter (OTC) in pharmacies.

I have already commented on the other posts of this blog the relevance of probiotics (“A new probiotic modulates microbiota against hepatocellular carcinoma” August 24th, 2016), as well as the microbiota that coexists with our body (“Bacteria in the gut controlling what we eat” October 12th, 2014; “The good bacteria of breast milk” February 3rd, 2013) and other animals (“Human skin microbiota … and our dog” December 25th, 2015; “The herbivore giant panda …. and its carnivore microbiota” September 30th, 2015).

Besides lactic acid bacteria and bifidobacteria, other microorganisms that are also used to a certain extent as probiotics are the yeast Saccharomyces cerevisiae, some strains of Escherichia coli, and some Bacillus, as we will see. Some clostridia are also used, related to what I commented in a previous post of this blog by March 21st, 2015 (“We have good clostridia in the gut ...”).

 

The Bacillus

In fact, Bacillus and clostridia have in common the ability to form endospores. And both groups are gram-positive bacteria, within the taxonomic phylum Firmicutes (Figure 1), which also includes lactic acid bacteria. However, bacilli (Bacillus and similar ones, but also Staphylococcus and Listeria) are more evolutionarily closer to lactobacillalles (lactic acid bacteria) than to clostridia ones. The main physiological difference between Clostridium and Bacillus is that the first are strict anaerobes while Bacillus are aerobic or facultative anaerobic.

Figure 1. Phylogenetic tree diagram of Gram-positive bacteria (Firmicutes and Actinobacteria). Own elaboration.

 

Bacterial endospores (Figure 2) are the most resistant biological structures, as they survive extreme harsh environments, such as UV and gamma radiation, dryness, lysozyme, high temperatures (they are the reference for thermal sterilization calculations), lack of nutrients and chemical disinfectants. They are found in the soil and in the water, where they can survive for very long periods of time.

Figure 2. Endospores (white parts) of Bacillus subtilis in formation (Image of Simon Cutting).

 

Bacillus in fermented foods, especially Asian

Several Bacillus are classically involved in food fermentation processes, especially due to their protease production capacity. During fermentation, this contributes to nutritional enrichment with amino acids resulting from enzymatic proteolysis.

Some of these foods are fermented rice flour noodles, typical of Thailand and Burma (nowadays officially Myanmar). It has been seen that a variety of microorganisms (lactic acid bacteria, yeasts and other fungi) are involved in this fermentation, but also aerobic bacteria such as B. subtilis. It has been found that their proteolytic activity digests and eliminates protein rice substrates that are allergenic, such as azocasein, and therefore they have a beneficial activity for the health of consumers (Phromraksa et al. 2009).

However, the best-known fermented foods with Bacillus are the alkaline fermented soybeans. As you know, soy (Glycine max) or soya beans are one of the most historically consumed nourishing vegetables, especially in Asian countries. From they are obtained “soy milk”, soybean meal, soybean oil, soybean concentrate, soy yogurt, tofu (soaked milk), and fermented products such as soy sauce, tempeh, miso and other ones. Most of them are made with the mushroom Rhizopus, whose growth is favoured by acidification or by direct inoculation of this fungus. On the other hand, if soy beans are left to ferment only with water, the predominant natural microbes fermenting soy are Bacillus, and in this way, among other things, the Korean “chongkukjang” is obtained, “Kinema” in India, the “thua nao” in northern Taiwan, the Chinese “douchi”, the “chine pepoke” from Burma, and the best known, the Japanese “natto” (Figure 3). Spontaneous fermentation with Bacillus gives ammonium as a by-product, and therefore is alkaline, which gives a smell not very good to many of these products. Nevertheless, natto is made with a selected strain of B. subtilis that gives a smoother and more pleasant smell (Chukeatirote 2015).

These foods are good from the nutritional point of view as they contain proteins, fibre, vitamins, and they are of vegetable or microbial origin. In addition, the advertising of the commercial natto emphasizes, besides being handmade and sold fresh (not frozen), its probiotic qualities, saying that B. subtilis (Figure 4) promotes health in gastrointestinal, immunologic, cardiovascular and osseous systems (www.nyrture.com). They say the taste and texture of natto are exquisite. It is eaten with rice or other ingredients and sauces, and also in the maki sushi. We must try it !

Figure 3. “Natto”, soybeans fermented with B. subtilis, in a typical Japanese breakfast with rice (Pinterest.com).

Figure 4. Coloured electronic micrograph of Bacillus subtilis (Nyrture.com).

 

Bacillus as probiotics

The endospores are the main advantage of Bacillus being used as probiotics, thanks to their thermal stability and to survive in the gastric conditions (Cutting 2011). Although Clostridium has also this advantage, its strict anaerobic condition makes its manipulation more complex, and moreover, for the “bad reputation” of this genus due to some well-known toxic species.

Unlike other probiotics such as Lactobacillus or Bifidobacterium, Bacillus endospores can be stored indefinitely without water. The commercial products are administered in doses of 10^9 spores per gram or per ml.

There are more and more commercial products of probiotics containing Bacillus, both for human consumption (Table 1) and for veterinary use (Table 2). In addition, there are also five specific products for aquaculture with several Bacillus, and also shrimp farms are often using products of human consumption (Cutting 2011).

For use in aquaculture, probiotic products of mixtures of Bacillus (B. thuringiensis, B. megaterium, B. polymixa, B. licheniformis and B. subtilis) have been obtained by isolating them from the bowel of the prawn Penaeus monodon infected with vibriosis. They have been selected based on nutrient biodegradation and the inhibitory capacity against the pathogen Vibrio harveyi (Vaseeharan & Ramasamy 2003). They are prepared freeze-dried or microencapsulated in sodium alginate, and it has been shown to significantly improve the growth and survival of shrimp (Nimrat et al., 2012).

As we see for human consumption products, almost half of the brands (10 of 25) are made in Vietnam. The use of probiotic Bacillus in this country is more developed than in any other, but the reasons are not clear. Curiously, as in other countries in Southeast Asia, there is no concept of dietary supplements and probiotics such as Bacillus are only sold as medications approved by the Ministry of Health. They are prescribed for rotavirus infection (childhood diarrhoea) or immune stimulation against poisoning, or are very commonly used as a therapy against enteric infections. However, it is not clear that clinical trials have been carried out, and they are easy-to-buy products (Cutting 2011).

 

Table 1. Commercial products of probiotics with Bacillus, for human consumption (modified from Cutting 2011).

Product	Country where it is made	Species of Bacillus
Bactisubtil ®	France	B. cereus
Bibactyl ®	Vietnam	B. subtilis
Bidisubtilis ®	Vietnam	B. cereus
Bio-Acimin ®	Vietnam	B. cereus and 2 other
Biobaby ®	Vietnam	B. subtilis and 2 other
Bio-Kult ®	United Kingdom	B. subtilis and 13 other
Biosporin ®	Ukraine	B. subtilis + B. licheniformis
Biosubtyl ®	Vietnam	B. cereus
Biosubtyl DL ®	Vietnam	B. subtilis and 1 other
Biosubtyl I and II ®	Vietnam	B. pumilus
Biovicerin ®	Brazil	B. cereus
Bispan ®	South Korea	B. polyfermenticus
Domuvar ®	Italy	B. clausii
Enterogermina ®	Italy	B. clausii
Flora-Balance ®	United States	B. laterosporus *
Ildong Biovita ®	Vietnam	B. subtilis and 2 other
Lactipan Plus ®	Italy	B. subtilis *
Lactospore ®	United States	B. coagulans *
Medilac-Vita ®	China	B. subtilis
Nature’s First Food ®	United States	42 strains, including 4 B.
Neolactoflorene ®	Italy	B. coagulans * and 2 other
Pastylbio ®	Vietnam	B. subtilis
Primal Defense ®	United States	B. subtilis
Subtyl ®	Vietnam	B. cereus
Sustenex ®	United States	B. coagulans

* Some labelled as Lactobacillus or other bacteria are really Bacillus

 

Table 2. Commercial products of probiotics with Bacillus, for veterinary use (modified from Cutting 2011).

Product	Animal	Country where it is made	Species of Bacillus
AlCare ®	Swine	Australia	B. licheniformis
BioGrow ®	Poultry, calves and swine	United Kingdom	B. licheniformis and B. subtilis
BioPlus 2B ®	Piglets, chickens, turkeys	Denmark	B. licheniformis and B. subtilis
Esporafeed Plus ®	Swine	Spain	B. cereus
Lactopure ®	Poultry, calves and swine	India	B. coagulans *
Neoferm BS 10 ®	Poultry, calves and swine	France	B. clausii
Toyocerin ®	Poultry, calves, rabbits and swine	Japan	B. cereus

 

The Bacillus species that we see in these Tables are those that really are found, once the identification is made, since many of these products are poorly labelled as Bacillus subtilis or even as Lactobacillus (Green et al. 1999; Hoa et al. 2000). These labelling errors can be troubling for the consumer, and especially for security issues, since some of the strains found are Bacillus cereus, which has been shown to be related with gastrointestinal infections, since some of them produce enterotoxins (Granum & Lund 1997; Hong et al. 2005)

The probiotic Bacillus have been isolated from various origins. For example, some B. subtilis have been isolated from the aforementioned Korean chongkukjang, which have good characteristics of resistance to the gastrointestinal tract (GI) conditions and they have antimicrobial activity against Listeria, Staphylococcus, Escherichia and even against B. cereus (Lee et al. 2017).

One of the more known probiotics pharmaceuticals is Enterogermina ® (Figure 5), with B. subtilis spores, which is recommended for the treatment of intestinal disorders associated with microbial alterations (Mazza 1994).

Figure 5. Enterogermina ® with spores of Bacillus subtilis (Cutting 2011)

 

Bacillus in the gastrointestinal tract: can they survive there ?

It has been discussed whether administered spores can germinate in the GI tract. Working with mice, Casula & Cutting (2002) have used modified B. subtilis, with a chimeric gene ftsH-lacZ, which is expressed only in vegetative cells, which can be detected by RT-PCR up to only 100 bacteria. In this way they have seen that the spores germinate in significant numbers in the jejunum and in the ileum. That is, spores could colonize the small intestine, albeit temporarily.

Similarly, Duc et al. (2004) have concluded that B. subtilis spores can germinate in the gut because after the oral treatment of mice, in the faeces are excreted more spores that the swallowed ones, a sign that they have been able to proliferate. They have also detected, through RT-PCR, mRNA of vegetative bacilli after spore administration, and in addition, it has been observed that the mouse generates an IgG response against bacterial vegetative cells. That is, spores would not be only temporary stagers, but they would germinate into vegetative cells, which would have an active interaction with the host cells or the microbiota, increasing the probiotic effect.

With all this, perhaps it would be necessary to consider many Bacillus as not allochthonous of the GI tract, but as bacteria with a bimodal growth and sporulation life cycle, both in the environment and in the GI tract of many animals (Hong et al. 2005).

Regarding the normal presence of Bacillus in the intestine, when the different microorganisms inhabiting the human GI tract are studied for metagenomic DNA analysis of the microbiota, the genus Bacillus does not appear (Xiao et al., 2015). As we can see (Figure 6), the most common are Bacteroides and Clostridium, followed by various enterobacteria and others, including bifidobacteria.

Figure 6. The 20 bacterial genera more abundant in the mice (left) and human (right) GI tract (Xiao et al. 2015).

 

In spite of this, several species of Bacillus have been isolated from the GI tract of chickens, treating faecal samples with heat and ethanol to select only the spores, followed by aerobic incubation (Barbosa et al. 2005). More specifically, the presence of B. subtilis in the human microbiota has been confirmed by selective isolation from biopsies of ileum and also from faecal samples (Hong et al. 2009). These strains of B. subtilis exhibited great diversity and had the ability to form biofilms, to sporulate in anaerobiosis and to secrete antimicrobials, thereby confirming the adaptation of these bacteria to the intestine. In this way, these bacteria can be considered intestinal commensals, and not only soil bacteria.

 

Security of Bacillus as probiotics

The oral consumption of important amounts of viable microorganisms that are not very usual in the GI treatment raises additional doubts about safety. Even more in the use of species that do not have a history of safe use in foods, as is the case of sporulated bacteria. Even normal bowel residents may sometimes act as opportunistic pathogens (Sanders et al. 2003).

With the exception of B. anthracis and B. cereus, the various species of Bacillus are generally not considered pathogenic. Of course, Bacillus spores are commonly consumed inadvertently with foods and in some fermented ones. Although Bacillus are recognized as GRAS for the production of enzymes, so far the FDA has not guaranteed the status of GRAS for any sporulated bacteria with application as a probiotic, neither Bacillus nor Clostridium. While Lactobacillus and Bifidobacterium have been the subject of numerous and rigorous tests of chronic and acute non-toxicity, and a lot of experts have reviewed data and have concluded that they are safe as probiotics, there is no toxicity data published on Bacillus in relation to their use as probiotics. When reviewing articles on Medline with the term “probiotic” and limited to clinical studies, 123 references appear, but Bacillus does not appear in any of them (Sanders et al. 2003).

Instead, there are some clinical studies where Bacillus strains have been detected as toxigenic. All this explains that some probiotic Bacillus producers refer to them with the misleading name of Lactobacillus sporogenes, a non-existent species, as can be seen from NCBI (https://www.ncbi.nlm.nih.gov/taxonomy/?term = Lactobacillus + sporogenes).

Finally, we should remember the joint report on probiotics of FAO (United Nations Food and Agriculture Organization) and WHO (World Health Organization) (FAO / WHO 2006), which suggests a set of Guidelines for a product to be used as a probiotic, alone or in the form of a new food supplement. These recommendations are:

1. The microorganism should be well characterized at the species level, using phenotypic and genotypic methods (e.g. 16S rRNA).
2. The strain in question should be deposited in an internationally recognized culture collection.
3. To evaluate the strain in vitro to determine the absence of virulence factors: it should not be cytotoxic neither invades epithelial cells, and not produce enterotoxins or haemolysins or lecithinases.
4. Determination of its antimicrobial activity, and the resistance profile, including the absence of resistance genes and the inability to transfer resistance factors.
5. Preclinical evaluation of its safety in animal models.
6. Confirmation in animals demonstrating its effectiveness.
7. Human evaluation (Phase I) of its safety.
8. Human evaluation (Phase II) of its effectiveness (if it does the expected effect) and efficiency (with minimal resources and minimum time).
9. Correct labelling of the product, including genus and species, precise dosage and conservation conditions.

Conclusions

The use of Bacillus as probiotics, especially in the form of dietary supplements, is increasing very rapidly. More and more scientific studies show their benefits, such as immune stimulation, antimicrobial activities and exclusive competition. Their main advantage is that they can be produced easily and that the final product, the spores, is very stable, which can easily be incorporated into daily food. In addition, there are studies that suggest that these bacteria may multiply in GI treatment and may be considered as temporary stagers (Cutting 2011).

On the other hand, it is necessary to ask for greater rigor in the selection and control of the Bacillus used, since some, if not well identified, could be cause of intestinal disorders. In any case, since the number of products sold as probiotics that contain the sporulated Bacillus is increasing a lot, one must not assume that all are safe and they must be evaluated on a case-by-case basis (Hong et al. 2005).

 

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