Category Archives: Lactic acid bacteria and products
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.
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.
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)
|GABA (gamma-amino-butyric acid)||Lactobacillus, Bifidobacterium|
|Norepinephrine||Escherichia, Bacillus, Saccharomyces|
|Serotonin||Candida, Streptococcus, Escherichia, Enterococcus|
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.
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
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.
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.
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.
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.
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.
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.
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.
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.
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?
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.
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).
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.
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.
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.
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
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
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
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