Category Archives: Lactic acid bacteria and products

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.

Fig 1 tree gram+ eng

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.

Fig 2 bacillus Simon Cutting

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 !

OLYMPUS DIGITAL CAMERA

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

Fig 4 Bs nyrture-com micrograf electro colorejada

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).

Figuresv1 copy.ppt

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.

Fig 6 Xiao nbt.3353-F2

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.

FAO WHO

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).

 

Bibliography

Barbosa TM, Serra CR, La Ragione RM, Woodward MJ, Henriques AO (2005) Screening for Bacillus isolates in the broiler gastrointestinal tract. Appl Environ Microbiol 71, 968-978.

Casula G, Cutting SM (2002) Bacillus probiotics: Spore germination in the gastrointestinal tract. Appl Environ Microbiol 68, 2344-2352.

Chukeatirote E (2015) Thua nao: Thai fermented soybean. J Ethnic Foods 2, 115-118.

Cutting SM (2011) Bacillus probiotics. Food Microbiol 28, 214-220.

Duc LH, Hong HA, Barbosa TM, Henriques AO, Cutting SM (2004) Characterization of Bacillus probiotics available for human use. Appl Environ Microbiol 70, 2161-2171.

FAO/WHO (2006) Probiotics in food. Health and nutritional properties and guidelines for evaluation. Fao Food and Nutrition Paper 85. Reports of Joint FAO/WHO expert consultations.

Fontana L, Bermudez-Brito M, Plaza-Diaz J, Muñoz-Quezada S, Gil A (2013) Sources, isolation, characterization and evaluation of probiotics. Brit J Nutrition 109, S35-S50.

Granum, P. E., T. Lund (1997) Bacillus cereus and its food poisoning toxins. FEMS Microbiol. Lett. 157:223–228.

Green, D. H., P. R. Wakeley, A. Page, A. Barnes, L. Baccigalupi, E. Ricca, S. M. Cutting (1999) Characterization of two Bacillus probiotics. Appl Environ Microbiol 65, 4288–4291.

Hoa, N. T., L. Baccigalupi, A. Huxham, A. Smertenko, P. H. Van, S. Ammendola, E. Ricca, A. S. Cutting (2000) Characterization of Bacillus species used for oral bacteriotherapy and bacterioprophylaxis of gastrointestinal disorders. Appl Environ Microbiol 66, 5241–5247.

Hong HA, Dic LH, Cutting SM (2005) The use of bacterial spore formers as probiotics. FEMS Microbiol Rev 29, 813-835.

Hong HA, Khaneja R, Tam NMK, Cazzato A, Tan S, Urdaci M, Brisson A, Gasbarrini A, Barnes I, Cutting SM (2009) Bacillus subtilis isolated from the human gastrointestinal tract. Res Microbiol 160, 134-143.

Lee S, Lee J, Jin YI, Jeong JC, Hyuk YH, Lee Y, Jeong Y, Kim M (2017) Probiotic characteristics of Bacillus strains isolated from Korean traditional soy sauce. LWT – Food Sci Technol 79, 518-524.

Mazza P (1994) The use of Bacillus subtilis as an antidiarrhoeal microorganism. Boll Chim. Farm. 133, 3-18.

Nimrat S, Suksawat S, Boonthai T, Vuthiphandchai V (2012) Potential Bacillus probiotics enhance bacterial numbers, water quality and growth during early development of white shrimp (Litopenaeus vannamei). Veterinary Microbiol 159, 443-450.

Phromraksa P, Nagano H, Kanamaru Y, Izumi H, Yamada C, Khamboonruang C (2009) Characterization of Bacillus subtilis isolated from Asian fermented foods. Food Sci Technol Res 15, 659-666.

Sanders ME, Morelli L, Tompkins TA (2003) Sporeformers as human probiotics: Bacillus, Sporolactobacillus, and Brevibacillus. Compr Rev Food Sci Food Safety 2, 101-110

Vaseeharan, B., P. Ramasamy (2003) Control of pathogenic Vibrio spp. by Bacillus subtilis BT23, a possible probiotic treatment for black tiger shrimp Penaeus monodon. Lett Appl Microbiol 36, 83–87

World Gastroenterology Organisation Global Guidelines (2011) Probiotics and Prebiotics.

Xiao et al. (2015) A catalogue of the mouse gut metagenome. Nature Biotechnol 33, 1103-1108.

Fig 0 pinterest-com cool bacillus-subtilis-science-comics

 

A new probiotic modulates gut microbiota against hepatocellular carcinoma

24th August 2016

Over the last years the beneficial effects of the human intestinal microbiota on various health markers have been displayed, such as inflammation, immune response, metabolic function and weight. The importance of these symbiotic bacteria of ours has been proved. You can see these other posts related with our microbiota: “The good clostridia avoid us from allergies“, “Gut bacteria controlling what we eat” or “Good bacteria of breast milk

At the same time it has been seen that probiotics can be a good solution for many diseases with affected gut microbiota. Indeed, the beneficial role of probiotics to reduce gastrointestinal inflammation and prevent colorectal cancer has been proven.

However, recently it has been found that probiotics may have beneficial effects in other parts of the body beyond the gastrointestinal tract, particularly with immunomodulatory effects on an hepatocellular carcinoma (HCC). In this way, researchers at the University of Hong Kong, along with other from University of Eastern Finland, have published a study (Li et al, PNAS, 2016), where they have seen reductions of 40% in weight and size of HCC liver tumours in mice which were administered with a new mixture of probiotics, “Prohep.”

Hepatocellular carcinoma (HCC) is the most common type of liver cancer is the 2nd most deadly cancers, and it is quite abundant in areas with high rates of hepatitis. In addition, sorafenib, the drug most widely used to reduce the proliferation of tumour, is very expensive. The cost of this multikinase inhibitor is €3400 for 112 tablets of 200 mg, the recommended treatment of four pills a day for a month. Instead, any treatment with probiotics that would proved to be effective and could replace this drug would be much cheaper.

The new probiotics mix Prohep consists of several bacteria: Lactobacillus rhamnosus GG (LGG), Escherichia coli Nissle 1917 (ECN) and the whole inactivated by heat VSL#3 (1: 1: 1) containing Streptococcus thermophilus, Bifidobacterium breve, Bf. longum, Bf. infantis, Lb. acidophilus, Lb. plantarum, Lb. paracasei and Lb. delbrueckii.

In the mentioned work, Li et al. (2016) fed mice with Prohep for a week before inoculating them with a liver tumour, and observed a 40% reduction in tumour weight and size in comparison to control animals. As shown in Figure 1, the effect was significant at 35 days, and also for those who were given the Prohep the same day of tumour inoculation. Obviously, the effect of tumour reduction was much more evident when the antitumour compound Cisplatin was administered.

These researchers saw that tumour reduction was due to the inhibition of angiogenesis. This is the process that generates new blood vessels from existing ones, something essential for tumour growth. In relation to the tumour reduction, high levels of GLUT-1 + hypoxic were found. That meant that there was hypoxia caused by the lower blood flow to the tumour, since this was 54% lower in comparison to controls.

 

Fig 1 Li-Fig1B tumor size - days tumor

Figure 1. Change in tumour size. ProPre: administration of Prohep one week before tumour inoculation; ProTreat: administration of Prohep the same day of tumour inoculation; Cisplatin: administration of this antitumoral. (Fig 1B from Li et al, 2016).

 

These authors also determined that there was a smaller amount of pro-inflammatory angiogenic factor IL-17 and of Th17 cells of the immune system, cells also associated with cancer. The lower inflammation and angiogenesis could limit the tumour growth.

Moreover, these researchers established that the beneficial effects of probiotics administration were associated with the abundance of beneficial bacteria in the mice gut microbiota, analysed by metagenomics. So, probiotics modulate microbiota, favouring some gut bacteria, which produce anti-inflammatory metabolites such as cytokine IL-10 and which suppress the Th17 cell differentiation.

 

Fig 2 gut microbiota Eye of Science

Figure 2. Bacteria of the human intestinal microbiota seen by scanning electron microscope (SEM) (coloured image of Eye of Science / Science Source)

 

Some of the bacteria identified by metagenomics in the microbiota of mice that were administered with Prohep were Prevotella and Oscillibacter. The first is a bacteroidal, gram-negative bacterium, which is abundant in the microbiota of rural African child with diets rich in carbohydrates. Oscillibacter is a gram-positive clostridial, known in humans as a producer of the neurotransmitter GABA. Both are an example of the importance of some clostridial and bacteroidals in the gut microbiota. In fact, they are majority there, and although they are not used as probiotics, are found increasingly more positive functions, such as avoiding allergies (see “The good clostridia avoid us from allergies“).

It is known that these bacteria produce anti-inflammatory metabolites and therefore they would be the main involved in regulating the activity of immune cells that cause tumour growth. The observed reduction of tumour in these experiments with mice would be the result of combined effect of these administered probiotic bacteria together with the microbiota itself favoured by them. We see a potential outline of these actions in Figure 3.

Fig 3 Sung fig 2

Figure 3. Simplified diagram of the possible mechanisms of gut bacteria influencing on the polarization of Th17 cells in the lamina propria of the intestinal mucosa. The microbiota bacteria activate dendritic cells, which secrete cytokines (IL-22, IL-23, IL-27). The bacteria can promote Th17 immunity inducing IL-23, which can be involved by means of TLR ligands signal or extracellular ATP or serum amyloid A (SAA). Meanwhile, some probiotic strains could inhibit the development of Th17 by means of the production of IL-12 and IL-27, in addition to promoting the growth and colonization of the bacteria that induce Th17 (Sung et al 2012, Fig. 2).

 

Although we know that the cancer progression is a very complex process and that in the tumour microenvironments there is an infiltration of many different types of immune system cells, such as T cells, neutrophils, killer cells, macrophages etc, the Th17 helper cell subpopulation appears to be prevailing in the tumour progression, and therefore these effects of probiotics and microbiota open good prospects.

It is still early to say whether these findings will contribute to the treatment of human liver cancer, and therefore research in humans is needed, in order to see if these probiotics could be used as such or in tandem with some drug, depending on the tumour stage and size. In any case, all this opens a new range of possibilities for research of the molecular mechanisms of the beneficial effects of probiotics beyond the intestinal tract.

 

Bibliography

El-Nezami H (2016 april 27) HKU develops novel probiotic mixture “Prohep” that may offer potential therapeutic effects on liver cancer. The University of Hong Kong (HKU) 27 Apr 2016

El-Nezamy H, Lee PY, Huang J, Sung YJ (2015) Method and compositions for treating cancer using probiotics. Patent WO 2015021936 A1

Li J, Sung CYJ, Lee N, Ni Y, Pihlajamäki J, Panagiotou G, El-Nezami H (2016) Probiotics modulated gut microbiota suppresses hepatocellular carcinoma growth in mice. PNAS E1306-E1315

Oelschlaeger TA (2010) Mechanisms of probiotic actions – A review. Int J Med Microbiol 300, 57-62

Packham C (2016) Probiotics dramatically modulate liver cancer growth in mice. Medical Press, Med Research 23 Feb 2016

Silgailis M (2016) Treating some cancers with probiotics in the future ? Probiotic Prohep. Lacto Bacto: Health, Microbes and More 23 Feb 2016

Sung CYJ, Lee NP, El-Nezami H (2012) Regulation of T helper by bacteria: an approach for the treatment of hepatocellular carcinoma. Int J Hepatology ID439024, doi:10.1155/2012/439024

UEF News and Events (2016) A novel probiotic mixture may offer potential therapeutic effects on hepatocellular carcinoma. University of Eastern Finland 1 Mar 2016

 

Bacteria of vineyard and terroir, and presence of Oenococcus in Priorat (South Catalonia) grapes

2nd May 2015 

The vine growers believe that the land on which they grow vines gives the wines a unique quality, and that is called terroir. We can consider that the physiological response of the vines to the type of soil and climatic conditions, together with the characteristics of the variety and form of cultivation, result in a wine organoleptic properties that define their terroir (Zarraonaindia et al 2015 ). However, it is not known if there could be a very specific microbiota of each terroir, as this subject has been barely studied.

Wine microorganisms in the grapes? Saccharomyces is not there or it has not been found there

The main protagonists of wine fermentations, alcoholic one (yeast Saccharomyces cerevisiae) and malolactic one (lactic acid bacteria Oenococcus oeni) usually do not appear until the must grape is fermenting to wine, in the cellar. In normal healthy grapes, S. cerevisiae is hardly found.

Oenococcus oeni in the grapes ? We have found it !

Regarding O. oeni, so far very little has been published about its presence and isolation from the grapes. In some works, as Sieiro et al (1990), or more recently Bae et al (2006), some lactic acid bacteria (LAB) have been isolated from the surface of grapes, but not O. oeni. Only Garijo et al (2011) were able to isolate a colony (only one) of O. oeni from Rioja grapes. Moreover, DNA of O. oeni has been detected in a sample of grapes from Bordeaux (Renouf et al 2005, Renouf et al 2007) by PCR-DGGE of rpoB gene, although in these works no Oenococcus has been isolated.

I am pleased to mention that recently our team have managed to isolate O. oeni from grapes, and typify them, and we are now working on a publication about it (Franquès et al 2015). Indeed, our research team of lactic acid bacteria (BL-URV), together with colleagues working on yeasts from the same group “Oenological Biotechnology” (Faculty of Oenology at the Universitat Rovira i Virgili in Tarragona, Catalonia, Spain) is working on a European project, called “Wildwine “(FP7-SME-2012 -315065), which aims to analyse the autochthonous microorganisms of Priorat area (South Catalonia), and select strains with oenological potential. This project also involves the Priorat Appellation Council and the cellar Ferrer-Bobet, as well as research groups and associations wineries from Bordeaux, Piedmont and Greece. In the framework of this project we took samples of grapes (Grenache and Carignan) from several vineyards of Priorat (Figure 1), as well as samples of wines doing malolactic fermentation. From all them we got 1900 isolates of LAB. We optimized isolation from grapes from the pulp and juice with various methods of enrichment, and so we got 110 isolated bacteria from grapes, identified as O. oeni by specific molecular techniques. Once typified, we have found that the molecular profiles of these strains do not coincide with commercial strains and so they are autochthonous. In addition, some of these strains from grapes were also found in the corresponding wine cellars.

Fig 1 garna-cari Priorat

Figure 1. Taking samples of Grenache (left) and Carignan (right) in Priorat area to isolate lactic acid bacteria such as Oenococcus (Pictures Albert Bordons).


The microbiota of grapes

The grapes have a complex microbial ecology, including yeasts, mycelial fungi and bacteria. Some are found only in grapes, such as parasitic fungi and environmental bacteria, and others have the ability to survive and grow in wines: especially yeasts, lactic acid bacteria (LAB) and acetic acid bacteria. The proportion of all them depends on the maturation of the grapes and the availability of nutrients.

When the fruits are intact, the predominant microbiota are basidiomycetous yeasts as Cryptococcus and Rhodotorula, but when they are more mature, they begin to have micro fissures that facilitate the availability of nutrients and explain the predominance just before the harvest of slightly fermentative ascomycetes as Candida, Hanseniaspora, Metschnikowia and Pichia. When the skin is already damaged more damaging yeasts may appear, as Zygosaccharomyces and Torulaspora, and acetic acid bacteria. Among the filamentous fungi occasionally there may have some very harmful as Botrytis (bunch rot) or Aspergillus producing ochratoxin. Although they are active only in the vineyard, their products can affect wine quality.

On the other hand, environmentally ubiquitous bacteria have been isolated from the grapes skin, as various Enterobacteriaceae, Bacillus and Staphylococcus, but none of them can grow in wine (Barata et al 2012).

Coming back to the possible specific microbiota of terroir, it has been found that some volatile compounds contributing to the aroma of the wine, such as 2-methyl butanoic acid and 3-methyl butanol, are produced by microorganisms isolated in the vineyards, as Gram-positive bacterium Paenibacillus, or the basidiomycetous fungus Sporobolomyces or the ascomycetous Aureobasidium. Therefore, there could be a relationship between some of the microbial species found in grapes and some detected aromas in wine, coming from the must of course (Verginer et al 2010).

Metagenomics as analytical tool of microbiota from grapes

Since conventional methods of isolation and cultivation of microorganisms are slow, laborious and some microbes cannot be grown up in the usual isolation media, massive sequencing methods or metagenomics are currently used. These consist of analysing all the DNA of a sample, and deducing which are the present microorganisms by comparing the sequences found with those of the databases. For bacteria the amplified DNA of V4 fragment from 16S RNA gene is used (Caporaso et al 2012).

This technique has been used with samples of botrytized wines (Bokulich et al 2012) and various LAB have been found (but not Oenococcus), including some not normally associated with wine. It has also been used to see the resident microbiota in wineries and how it changes with the seasons, resulting that in the surfaces of tanks and machinery of the cellar there is a majority of microorganisms neither related with wine nor harmful (Bokulich et al 2013).

With this technique Bokulich et al (2014) have also analysed the grapes and they have seen clear differences between the proportions of bacterial groups (and fungi) from different places, different varieties, as well as environmental or bio geographical conditions. For example, when analysing 273 samples of grape musts from California, the 3 varieties (Cabernet, Chardonnay and Zinfandel) are quite discriminated in a principal components analysis with respect to the bacterial communities found in each sample (Figure 2).

Thus, the dominant bacterial taxa or groups in a variety or given environment could provide some specifics traits on those wines, and this could explain some regional or terroir patterns in the organoleptic properties of these wines (Bokulich et al 2014).

Fig 2 ACP Bokulich 2014

Figure 2. Principal component analysis of bacterial communities of grape musts samples of Sonoma (California) from 3 varieties (Cabernet in red, Chardonnay in green and Zinfandel in blue) (Bokulich et al 2014).


We have also carried out a massive sequencing study with the same grape samples from which we have obtained isolates of O. oeni, as said before (Franquès et al 2015), and in more than 600,000 analysed sequences of 16S rRNA, we have found mainly Proteobacteria and Firmicutes. Among these gram-positive, we have found sequences of lactic acid bacteria (15%) and from these we have successfully confirmed the presence of O. oeni in 5% of the sequences. Therefore, we have isolated O. oeni from grapes and we have detected their DNA in the samples.

The bacterial microbiota of the vineyards and soil

As we see, microbiota of grapes and wine has been studied a little, but the soil microbiota has not been characterized. This one can define more clearly the terroir, which is influenced by the local climate and characteristics of the vineyard.

In Figure 3 the main genera found in different parts of the vine and soil are summarized (Gilbert et al 2014).

Fig 3 Gilbert 2014

Figure 3. Main bacteria and fungi associated with organs and soil of Vitis vinifera (Gilbert et al 2014)


Recently an interesting scientific work (Zarraonaindia et al 2015) has been published on this subject, with the aim to see if the soil could be the main original source of bacteria that colonize the grapes. These authors took samples of soil, roots, leaves, flowers and grapes from Merlot vines, from different areas and years, of Suffolk, New York, and they analysed the bacterial DNA by 16S rRNA sequencing. They found that 40% of the species found were present in all samples of soil and roots, while there was more variability in leaves and fruits, and moreover, 40% of those found in leaves and fruits were also found in soils. All this suggests that many bacteria originate in the soil.

Regarding the type of bacteria, they found that Proteobacteria (especially Pseudomonas and Methylobacterium) predominated (Figure 4), mainly in the aerial parts of the plant. There were also Firmicutes as expected, and Acidobacteria and Bacteroides.

Fig 4 microbiota vineyard

Figure 4. Composition of the bacterial community, at Phylum level, in samples from different organs of the vine and its soil (Zarraonaindia et al 2015).


Although variations were observed in all samples depending on the year (there may be different climatic conditions) and according to different edaphic factors (pH, C: N, humidity), the principal-components analysis (Figure 5) showed that the main types of samples (soil, roots, leaves, grapes) differ quite well, and bacterial taxon composition in samples of grape juice before fermentation is similar to that of grapes.

Fig 5 distribució grups mostres OTUs

Figure 5. Principal-components analysis showing the similarities in terms of the composition of bacterial taxonomic groups, among sample types, including musts (Zarraonaindia et al 2015).


This suggests that the bacterial community found in grapes remains relatively stable until the processing to musts, and that it is more stable than the differences between organs. At the same time, a large number of representatives of bacterial phyla of the grapes come from the soil. This can be explained because when grapes are harvested by hand, they are often placed in boxes that are left on the ground, or for mechanical harvest, the machinery used removes the soil and generates dust, which can colonize the grapes.

Therefore, the soil microbiota is a source of bacteria associated with vines and may play a role in the must and therefore in the wine, and potentially in the formation of the terroir characteristics. Some of these bacteria may have some roles not yet known in productivity or disease resistance of the plant, or contribute to the organoleptic characteristics of wine (Zarraonaindia et al 2015).

In addition, and thinking in wine microorganisms responsible for fermentations, as said, in our laboratory we have confirmed that there are some O. oeni strains in grapes and we have confirmed this by detecting their DNA in the same grapes.

References

Bae S, Fleet GH, Heard GM (2006) Lactic acid bacteria associated with wine grapes from several Australian vineyards. J Appl Microbiol 100, 712-727

Barata A, Malfeito-Ferreira M, Loureiro V (2012) The microbial ecology of wine grapes (Review). Int J Food Microbiol 153, 243-259

Bokulich NA, Joseph CML, Allen G, Benson AK, Mills DA (2012) Next-generation sequencing reveals significant bacterial diversity of botrytized wine. Plos One 7, e36357

Bokulich NA, Ohta M, Richardson PM, Mills DA (2013) Monitoring seasonal changes in winery-resident microbiota. Plos One 8, e66437

Bokulich NA, Thorngate JH, Richardson PM, Mills DA (2014) Microbial biogeography of wine grapes is conditioned by cultivar, vintage, and climate. PNAS nov 25, E139-E148

Caporaso JG, Lauber CL, Walters WA, Berg-Lyons D, Huntley J, Fierer N, Owens SM, Betley J, Fraser L, Bauer M, Gormley N, Gilbert JA, Smith G, Knight R (2012) Ultra-high-throughput microbial community analysis on the Illumina HiSeq and MiSeq platforms. ISME J 6, 1621–1624

Franquès J, Araque I, Portillo C, Reguant C, Bordons A (2015) Presence of autochthonous Oenococcus oeni in grapes and wines of Priorat in South Catalonia. Article in elaboration.

Garijo P, López R, Santamaría P, Ocón E, Olarte C, Sanz S, Gutiérrez AR (2011) Eur Food Res Technol 233, 359-365

Gilbert JA, van der Lelie D, Zarraonaindia I (2014) Microbial terroir for wine grapes. PNAS 111, 5-6

Renouf V, Claisse O, Lonvaud-Funel A (2005) Understanding the microbial ecosystem on the grape berry surface through numeration and identification of yeast and bacteria. Aust J Grape Wine Res 11, 316-327

Renouf V, Claisse O, Lonvaud-Funel A (2007) Inventory and monitoring of wine microbial consortia. Appl Microbiol Biotechnol 75, 149-164

Sieiro C, Cansado J, Agrelo D, Velázquez JB, Villa TG (1990) Isolation and enological characterization of malolactic bacteria from the vineyards of North-western Spain. Appl Environ Microbiol 56, 2936-2938

Verginer M, Leitner E, Berg G (2010) Production pf volatile metabolites by grape-associated microorganisms. J Agric Food Chem 58, 8344-8350

Zarraonaindia I, Owens SM, Weisenhorn P, West K, Hampton-Marcell J, Lax S, Bokulich NA, Mills DA, Martin G, Taghavi S, Van der Lelie D, Gilbert JA (2015) The soil microbiome influences grapevine-associated microbiota. mBio 6, e02527-14

Bacteria in the gut are controlling what we eat

It seems to be so: the microbes in our gastrointestinal tract (GIT) influence our choice of food. No wonder: microbes, primarily bacteria, are present in significant amounts in GIT, more than 10 bacterial cells for each of our cells, a total of 1014 (The human body has about 1013 cells). This amounts to about 1-1.5 kg. And these bacteria have lived with us always, since all mammals have them. So, they have evolved with our ancestors and therefore they are well suited to our internal environment. Being our bodies their habitat, much the better if they can control what reaches the intestine. And how can they do? Then giving orders to the brain to eat such a thing or that other, appropriate for them, the microbes.

Imagen1Figure 1.Command centre of the gastrointestinal tract” (own assembly,  Albert Bordons)

Well, gone seriously, there is some previous work in this direction. It seems there is a relationship between preferences for a particular diet and microbial composition of GIT (Norris et al 2013). In fact, it is a two-way interaction, one of the many aspects of symbiotic mutualism between us and our microbiota (Dethlefsen et al 2007).

There is much evidence that diet influences the microbiota. One of the most striking examples is that African children fed almost exclusively in sorghum have more cellulolytic microbes than other children (De Filippo et al 2010).

The brain can also indirectly influence the gut microbiota by changes in intestinal motility, secretion and permeability, or directly releasing specific molecules to the gut digestive lumen from the sub epithelial cells (neurons or from the immune system) (Rhee et al 2009).

The GIT is a complex ecosystem where different species of bacteria and other microorganisms must compete and cooperate among themselves and with the host cells. The food ingested by the host (human or other mammal) is an important factor in the continuous selection of these microbes and the nature of food is often determined by the preferences of the host. Those bacteria that are able to manipulate these preferences will have advantages over those that are not (Norris et al 2013).

Recently Alcock et al (2014) have reviewed the evidences of all this. Microbes can manipulate the feeding behaviour of the host in their own benefit through various possible strategies. We’ll see some examples in relation to the scheme of Figure 2.

 

Fig 2 human microbiome behaviour appetite

Figure 2. As if microbes were puppeteers and we humans were the puppets, microbes can control what we eat by a number of marked mechanisms. Adapted from Alcock et al 2014.

 

People who have “desires” of chocolate have different microbial metabolites in urine from people indifferent to chocolate, despite having the same diet.

Dysphoria, id est, human discomfort until we eat food which improve microbial “welfare”, may be due to the expression of bacterial virulence genes and perception of pain by the host. This is because the production of toxins is often triggered by a low concentration of nutrients limiting growth. The detection of sugars and other nutrients regulates virulence and growth of various microbes. These directly injure the intestinal epithelium when nutrients are absent. According to this hypothesis, it has been shown that bacterial virulence proteins activate pain receptors. It has been shown that fasting in mice increases the perception of pain by a mechanism of vagal nerve.

Microbes can also alter food preferences of guests changing the expression of taste receptors on the host. In this sense, for instance germ-free mice prefer more sweet food and have a greater number of sweet receptors on the tongue and intestine that mice with a normal microbiota.

The feeding behaviour of the host can also be manipulated by microbes through the nervous system, through the vagus nerve, which connects the 100 million neurons of the enteric nervous system from the gut to the brain via the medulla. Enteric nerves have receptors that react to the presence of certain bacteria and bacterial metabolites such as short chain fatty acids. The vagus nerve regulates eating behaviour and body weight. It has been seen that the activity of the vagus nerve of rats stimulated with norepinephrine causes that they keep eating despite being satiated. This suggests that GIT microbes produce neurotransmitters that can contribute to overeating.

Neurotransmitters produced by microbes are analogue compounds to mammalian hormones related to mood and behaviour. More than 50% of dopamine and most of serotonin in the body have an intestinal origin. Many persistent and transient inhabitants of the gut, including E. coli, several Bacillus, Staphylococcus and Proteus secrete dopamine. In Table 1 we can see the various neurotransmitters produced by GIT microbes. At the same time, it is known that host enzymes such as amine oxidase can degrade neurotransmitters produced by microorganisms, which demonstrates the evolutionary interactions between microbes and hosts.

 

Table 1. Diversity of neurotransmitters isolated from several microbial species (Roschchina 2010)

Neurotransmitter Genera
GABA (gamma-amino-butyric acid) Lactobacillus, Bifidobacterium
Norepinephrine Escherichia, Bacillus, Saccharomyces
Serotonin Candida, Streptococcus, Escherichia, Enterococcus
Dopamine Bacillus, Serratia
Acetylcholine Lactobacillus

 

Some bacteria induce hosts to provide their favourite nutrients. For example, Bacteroides thetaiotaomicron inhabits the intestinal mucus, where it feeds on oligosaccharides secreted by goblet cells of the intestine, and this bacterium induces its host mammal to increase the secretion of these oligosaccharides. Instead, Faecalibacterium prausnitzii, a not degrading mucus, which is associated with B. thetaiotaomicron, inhibits the mucus production. Therefore, this is an ecosystem with multiple agents that interact with each other and with the host.

As microbiota is easily manipulated by prebiotics, probiotics, antibiotics, faecal transplants, and changes in diet, controlling and altering our microbiota provides a viable method to the otherwise insoluble problems of obesity and poor diet.

 

References

Alcock J, Maley CC, Aktipis CA (2014) Is eating behavior manipulated by the gastrointestinal microbiota? Evolutionary pressures and potential mechanisms. BioEssays 36, DOI: 10.1002/bies.201400071

De Filippo C, Cavalieri D, Di Paola M, Ramazzotti M, et al (2010) Impact of diet in shaping gut microbiota revealed by a comparative study in children from Europe and rural Africa. Proc Natl Acad Sci USA 107:14691–6

Dethlefsen L, McFall-Ngai M, Relman DA (2007) An ecological and evolutionary perspective on human-microbe mutualism and disease. Nature 449:811-818

Lyte M (2011) Probiotics function mechanistically as delivery for neuroactive compounds: Microbial endocrinology in teh design and use of probiotics. BioEssays 33:574-581

Norris V, Molina F, Gewirtz AT (2013) Hypothesis: bacteria control host appetites. J Bacteriol 195:411–416

Rhee SH, Pothoulakis C, Mayer EA (2009) Principles and clinical implications of the brain–gut–enteric microbiota axis. Nature Reviews Gastroenterology and Hepatology 6:306-314

Roschchina VV (2010) Evolutionary considerations of neurotransmitters in microbial, plant, and animal cells. In Lyte M, Freestone PPE, eds; Microbial Endocrinology: Interkingdom Signaling in Infectious Disease and Health. New York: Springer. pp. 17–52

The good bacteria of breast milk

Breast milk, besides being very nutritious, provides bioactive constituents that favor the development of the infant immune system and prevent diseases. From this point of view, the best known compounds are maternal immunoglobulins, immunocompetent cells and various antimicrobials. It also contains prebiotic substances, ie, several molecules such as oligosaccharides, which stimulate the growth of specific bacteria in the gut of the child.

However, other important constituents of breast milk, unsuspected until few years ago, are the bacteria. In fact, milk is not sterile, it contains microorganisms, primarily beneficial bacteria that help to establish the intestinal microbiota of the newborn, and which are the first to settle there. Although artificial milk are made to resemble the breast milk, they remain distinct and do not contain bacteria. And for this reason, the intestinal microbiota of breast-fed infants is different than those fed with artificial breast milk.

 

1-BL-mamant

Lactobacilli (image from AJC1Flickr) and suckling baby (© Photos.com)

Just a few weeks ago was published a work ( Cabrera-Rubio et al., 2012 ) in the American Journal of Clinical Nutrition that had a good coverage in media, blogs and networks ( click here for an example), because it shows the great diversity of bacteria present in the breast milk.

Although this work done by Valencian researchers (Cavanilles Institute, University of Valencia and CSIC-IATA) with Finnish researchers is not the first study that examines this issue, this study shows that bacteria are from very diverse species.

One of the novelties of this paper is the method used, taking advantage of the latest molecular biology: they studied the microbiome in breast milk, that is, the analysis of all possible bacteria present in the samples, by DNA sequencing, without the traditional isolation of living bacteria in plates. To do so, from the aseptically collected milk, DNA is extracted and the gene fragments of bacterial 16S rRNA are amplified by PCR. These amplified genes are sequenced by pyrosequencing (454 Roche GS-FLX), the most innovative and rapid sequencing technology: a machine of this allows about 400 million base pairs (bp) of DNA in 10 hours. From the rRNA gene of each possible bacteria some 500 bp are sequenced. Thus, in this study about 120,000 sequences have been analyzed, corresponding to 2600 sequences per milk sample.

By comparing these sequences with the databases and applying statistical methods conclusions can be drawn on what taxonomic groups (genera and species) bacteria are present and in what proportion.

 

2-Cabrera2012 generes bacteris

Predominant genera of bacteria in breast milk (Cabrera-Rubio et al., 2012)

As shown in the figure above, Cabrera et al. found in the milk of healthy mothers that the predominant genera are Leuconostoc, Weissella, Lactococcus and Staphylococcus, of which the first three are lactic acid bacteria. Although these are predominant in colostrum and milk during the first months, then other bacteria are increasing their numbers, such as Veillonella Leptotrichia (anaerobic gram-negative bacteria), which are typical commensal of the oral cavity. In total, about 1000 species have been found, that vary depending on the mother. Curiously, there are significant variations on whether delivery had been vaginal or cesarean, and on the obesity of the mother. The reasons for this are not yet clear.

And where the bacteria in breast milk come from ?

Besides the identifications made in this study of Cabrera et al. (2012) on the basis of DNA present, it has been observed by making viable counts that the total number of bacteria in breast milk is between 2·104 and 3·105 per ml (Juan Miguel Rodríguez), that is, a quantity not negligible . What is its origin?

The study of the microbiome of Cabrera et al. also concluded that the composition of different bacteria is somewhat different from that of other bacterial communities in the human body (the human bacterial niches: skin, mouth, digestive system, vagina, etc), and therefore the milk microbiome is not a particular subset of one of these niches.

The group Probilac from Universidad Complutense de Madrid,  whose head is Juan Miguel Rodriguez, a friend and colleague of Red BAL (Spanish network of lactic acid bacteria) is working in this area for years (ex: Martin et al 2003 , Martin et al 2004).

As discussed in a recent review published by this group (Fernández et al 2012), the bacteria present in the breast milk would come from three possible sources (figure below): skin bacteria from the same breast, the oral cavity of the infant, and the most surprising, commensal bacteria of the maternal gut that pass to milk by the entero-mammary pathway.

 

3-fig Fdez Review

Potential sources of bacteria present in human colostrum and milk, including the transit of intestinal commensal bacteria to the milk by the entero-mammary pathway (Fernández et al., 2012). DC: dendritic cells.

Indeed, several studies had shown that dendritic cells cross the intestinal epithelium (between enterocytes) and may take commensal bacteria of the gut lumen, incorporating them by endocytosis, but keeping them alive. See details in the following diagram.

 

4-JM Rodríguez dendritic LAB no lege

Dendritic cell capturing gut bacteria (Scheme of J.M. Rodríguez, group Probilac, Univ. Complutense de Madrid).

These dendritic cells travel through the circulatory system, reaching the mammary glands, where it seems that include bacteria to milk. This is the the entero-mammary pathway.

In this breast microbiota, bacteria from breast skin and from oral cavity of the child also would be incorporated. Some of these bacteria the child’s oral cavity are actually related to those of its gastrointestinal tract. As the first bacteria inhabiting this tract are those of the vaginal microbiota during birth (and intestinal if delivery is cesarean), this would explain the phylogeny of certain bacteria in the milk of these microbiota.

In summary, we see as the “good” bacteria (lactic acid bacteria, but also bifidobacteria and other) from maternal gut, by different ways, arrive to breast milk, and the reach the child’s gut, developing there the child’s microbiota, and helping to complete the neonatal immune system.

Bibliography

Cabrera-Rubio R, MC Collado, K Laitinen, S Salminen, E Isolauri, A Mira (2012) The human milk microbiome changes over lactation and is shaped by maternal weight and mode of delivery. American J Clinical Nutrition 96, 544–51

Grupo Probilac (Juan Miguel Rodríguez Gómez) Microbiota de la leche humana en condiciones fisiológicas: http://www.ucm.es/info/probilac/microbiota2.htm, Departamento de Nutrición, Bromatología y Tecnología de los Alimentos, Facultad de Veterinaria, Universidad Complutense de Madrid

Fernández L, S Langa, V Martín, A Maldonado, E Jiménez, R Martín, JM Rodríguez (2012) The human milk microbiota: Origin and potential roles in health and disease. Pharmacological Research http://dx.doi.org/10.1016/j.phrs.2012.09.001

Hunt KM JA Foster, LJ Forney, UME Schütte, DL Beck, Z Abdo, LK Fox, JE Williams, MK McGuire, MA McGuire (2011) Characterization of the diversity and temporal stability of bacterial communities in human milk. PLoS ONE 6:e21313.

Martín R, S Langa, C Revriego, E Jiménez, ML Marín, J Xaus, L Fernández, JM Rodríguez (2003) Human milk is a source of lactic acid bacteria for the infant gut. J Ped. 143, 754-758.

Martín R, S Langa, C reviriego, E Jiménez, ML Marín, M Olivares, J Boza, J Jiménez, L fernández, J Xaus, JM Rodríguez (2004) The commensal microflora of human milk: new perspectives for food bacteriotherapy and probiotics. Trends Food Sci Technol 15:121–7.

Other references

Adlerberth I (2006) Reduced enterobacterial and increased staphylococcal colonization of the infantile bowel: an effect of hygienic lifestyle. Pediatric Res 59, 96-101.

Albesharata R et al (2011) Phenotypic and genotypic analyses of lactic acid bacteria in local fermented food, breast milk and faeces of mothers and their babies. Syst App Microb 34, 148–155

Domínguez-Bello MG et al (2010) Delivery mode shapes the acquisition and structure of the initial microbiota across multiple body habitats in newborns. Proc Natl Acad Sci USA;107:11971–5.

Huurre A et al (2008) Mode of delivery—effects on gut microbiota and humoral immunity. Neonatology;93:236–40

LeBouder E et al (2006) Modulation of neonatal microbial recognition: TLRmediated innate immune responses are specifically and differentially modulated by human milk. J Immunol;176:3742–52.

Martín R et al (2009) Isolation of bifidobacteria from breast milk and assessment of the bifidobacterial population by PCR-denaturing gradient gel electrophoresis and quantitative real-time PCR. Appl Environ Microbiol 75:965–9.

Pérez PF et al (2007) Bacterial imprinting of the neonatal immune system: lessons from maternal cells? Pediatrics 119: 724–732.

Rescigno M et al (2001) Dendritic cells shuttle microbes across gut epithelial monolayers. Immunobiology 204:572–81.

Stockinger S et al (2001) Establishment of intestinal homeostasis during the neonatal period. Cell Mol Life Sci;68: 3699–712.

European cheese from 7400 years ago, and “yoghourt” in the Sahara 7000 years ago

One month ago (12th December 2012) a work (Salque et al. 2012) was published online in Nature, which provides archaeological evidence of cheese making in present Poland about 5400 years before Christ (BC). And in last June was also published another study in Nature (Dunne et al. 2012) which evidenced the production of fermented milk products like yoghourt in northeastern Sahara (now Libya) about 5000 BC.

Using the milk of other animals

Agriculture, that is, the domestication of plants by humans, began between 10000 and 5000 BC mainly in the Middle East (the Fertile Crescent, from the Nile to the Euphrates), but also independently in other regions, such as India, China and various parts of America and Africa. The Neolithic agricultural revolution led to the establishment of sedentary populations and the subsequent birth of cities and civilizations. At the same time, in these sites there were domesticated also animals, but it is likely that the domestication of cattle, sheep and goats have already occurred before, in nomad populations. The use of these animals brought some important advances, using them for secondary uses without killing them (the primary use is the flesh) such as traction, wool, and milk and dairy products.

1-M Kiani Qashqai Persia 3-NOMAD-PLACE-milk

Nomad Qashqai (Persia) milking a sheep. Photo: M. Kiani

The first pictorial and written records of the use of the milk of domestic animals are from Egypt and Mesopotamia around 3000 BC. But recently, the first clear evidence for previous organic waste stored in ceramic remains has been found, by analyzing the values of δ 13C (ratio between the isotopes 13C and 12C) of the main fatty acids from fat of milk. This technique, from Dudd and Eversheds (1998), is based on the differences between the values of δ 13C of stearic acid (C 18:0) in milk and adipose tissue of the same body of animal, due to the higher proportion of carbon derived from carbohydrates in the diet used in the biosynthesis of C 18:0 in body fat compared to milk, where 40% of C 18:0 derived from unsaturated fats.

This technique of δ 13C has shown the use of milk in the 4th millennium BC in Britain, in the 6th millennium in Eastern Europe, and recently (Eversheds et al. 2008) has been shown that in the 7th millennium BC, 9000 years ago, there was milking in the Middle East and Southeast Europe, particularly in Anatolia.

But when the adults began to drink human milk?

2-beure llet

As you know, the lactose of milk is not tolerated by many adults, especially of Asian, Native Americans and many Africans. The enzyme lactase that hydrolyzes lactose into glucose and galactose is present in all the babies, but like all other mammals, when they become older, the gene for lactase is not expressed. The exception is those people that maintain the production of lactase in adulthood and so they can drink milk without problems. For those who do not tolerate milk, the reason is due to lactose fermentation by bacteria in the gut, which gives rise to diarrhea, flatulence and other disorders.

3-Rainer Zenz 550px-Laktoseintoleranz-1.svg

Percentages of human populations not tolerant to lactose. Map made by Rainer Zenz.

The humans more tolerant to lactose are of European origin and those in regions nearby the Sahara and the Middle East. In Europe there is a gradient from high to low tolerance northwest towards the southeast. Molecular biology studies have shown that tolerance to lactose appeared by mutation of a single nucleotide at different times and places, between 8000 and 3000 years ago, in pastoral peoples of northern Europe and Arabia (Swallow 2003, Enattah et al. 2008, Tellam 2012). This genetic characteristic was selected due to its positive nutritional benefits, and also because in the desert milk is a source of water, and also in northern Europe milk can replace the lack of calcium due to low solar radiation and therefore short synthesis of vitamin D needed for calcium absorption.

Cheese and fermented milk products for lactose intolerants

Cheese is the curdled milk from which is extracted, in part or all, the whey, id est, the milk with water soluble components, which are mostly lactose. The remaining precipitate is the cheese, which contains fat and milk protein but very little lactose. Therefore, for people lactose-intolerant cheese is a food nutritionally equivalent to milk, but without the inconvenience of lactose. In addition, cheese is kept longer than milk and takes many different tastes and textures, depending on the curdling process and on microorganisms involved in their maturation. In fermented milk such as yoghourt and other (Kefir, Kumi, Leben, etc.) microorganisms are involved, especially lactic acid bacteria, which consume part of lactose and produce lactic acid, which favors conservation. The content of lactose in these fermented milks is not as low as in cheese but they can be consumed by most lactose intolerant people.

For this reason, the use of various types of cheese and / or fermented milk is almost universal to humans, regardless of whether or not they are tolerant to lactose and probably it existed in various nomad peoples, with the first domesticated animals, and surely this was the first way to use the milk of these animals.

Evidence of cheese made in Europe about 7400 years

As said earlier, a work (Salque et al 2012) has been recently published online in Nature which provides archaeological evidence of cheese making in today’s Poland about 5400 years before Christ (BC).

At the beginning of the Neolithic sites (about 8000 years) from various parts of Europe containers with small holes appear, with shaped sieve, that have been thought for years as strainers cheese, similar to those used today in some regions. The milk is placed in the container, the rennet is added (from the stomach of ruminants, containing protease), and the precipitated curdled is squeezed, separating out the whey through the holes, to get the cheese (Subbaraman 2012).

4-ceramica formtagera3

Drawing representing a reconstructed vessel (left) and a portion of an actual piece of this container (right) with holes as a sieve, from a site of Kuyavia region (in central Poland). Image from Salque et al. (2012).

Salque et al. (2012) have shown by the above mentioned technique δ 13C (in addition to analyze by gas chromatography the composition of lipids) that the remains of fatty acids found at the site of vessel Kuyavia (north of Warsaw) were coming from milk. The fat composition and δ 13C values ​​of these vessels strainers are different from those found in other containers like pots where probably meat of different animals was cooked. Therefore, they demonstrate that these containers were used to make cheeses strainers, specifically about 7400 years ago. The authors emphasize the importance of this type of pottery in the processing of dairy products, indicating in particular the importance for lactose-intolerant prehistoric communities.

Evidence of fermented milk (yoghourt ?) in the Sahara 7000 years ago

As said above, last June another study was published in Nature (Dunne et al. 2012), which evidenced the production of fermented milk in northeastern Sahara (now Libya) about 7000 years ago.

In contrast to the well known process of early Neolithic settlements and agriculture in the Middle East, in the Sahara the pastoralism with cows, sheep and goats began long before the domestication of plants. Seeing the present desert of Sahara, so arid and inhospitable, it seems impossible that human communities prospered there with large herds, but this region enjoyed a very favorable climatically wet period that began some 10.000 years ago and there is plenty of evidence that 8000 years ago there proliferated all types of wildlife in the savannas of the current Sahara. Groups of hunters and gatherers who lived there already used the pottery to preserve food, and gradually, with the increase of the drought, had become more dependent on livestock.

A demonstration of these nomad livestock are the remarkable paintings and rock carvings found in the desert of southwest Libya (Wadi Teshuinat or Takarkori Acacus mountains, or in the area of Wadi Tiksatin Messak) from some 7000 years ago, possibly the most important concentration of prehistoric art in the world, with many scenes of daily life. In these representations it can be seen the importance of livestock for these humans, with drawings of obvious milking cows. However, there is no reliable dating of these prints.

5-pintures rupestres Sahara

Schematic drawings of Wadi Teshuinat cave, southwest Libya. Figure taken from Dunne et al. (2012).

The group of Julie Dunne and Richard Eversheds at the University of Bristol with the group of Savino di Lernia from University of Sapienza, studied the remains of fat present in the pottery of Takarkori site by gas chromatography coupled with mass spectrometry, and the aforementioned technical isotopes (δ 13C). Their results show that these potteries were used to produce fermented milk products like yoghourt, between 7000 and 4800 years ago. In addition, they found that milk fat came from a variety of plants from different places, which suggests that people were migrating with their herds, depending on the season. This work confirms that the economy of dairy products derived from domesticated cattle was active during this period, probably to compensate for lactose intolerant.

6-vaso_murzuq

Murzuq ceramics, from the site of Takarkori, Libya. Photo: Savino di Lernia

Following this work, some scientists (Callaway 2012) have suggested that subsequent to this period, the lactose tolerance mutation arose in Europe and Arabia and spread through North Africa due to its advantages. In the increasingly arid climate of the desert, to drink fresh and uncontaminated milk should lead to a better hydration respect to other people which had the tolerance gene not activated. Thus, there was a strong selective pressure for the spread of lactose tolerance in north Africa.

 

Bibliography

Arjamaa O, T Vuorisalo (2010) Genes, cultura y dieta. Investigación y Ciencia, 405, june 2010, 69-77

Callaway E (2012) Pottery shards put a date on Africa’s dairying. North Africans may have been making yoghurt 7,000 years ago. Nature News, 20 june 2012-12-22

Dudd SN, RP Evershed (1998) Direct Demonstration of Milk as an Element of Archaeological Economies. Science 282, 1478-1481

Dunne J et al (2012) First dairying in green Saharan Africa in the fifth millennium bc. Nature 486, 390–394 (21 June 2012) doi:10.1038/nature11186

Enattah NS et al. (2008) Independent introduction of two lactase-persistence alleles into human populations reflects different history of adaptation to milk culture. Am J Human Genetics 82, 57-72.

Evershed RPet al. (2008) Earliest date for milk use in the Near East and southeastern Europe linked to cattle herding. Nature 455, 528-531 (25 September 2008), doi:10.1038/nature07180

Salque, M. et al. (2012) Earliest evidence for cheese making in the sixth millennium BC in northern Europe. Nature http://dx.doi.org/10.1038/nature11698

Subbaraman, N (2012) Art of cheese-making is 7,500 years old. Neolithic pottery fragments from Europe reveal traces of milk fats. Nature News, 12 dec 2012

Swallow DM (2003) Genetics of lactase persistence and lactose intolerance. Annu. Rev. Genet. 37, 197-219

Tellam R (2012) How dairying shaped the human genome. International Milk Genomics Consortium

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