Patent Application
20220095648
The present invention is in the field of nutrition, and more particularly human and animal nutrition. It relates to the use of a Thraustochytrid biomass for maintaining gut barrier function in an individual.
In animal production, several factors during the rearing period are likely to influence the preservation of the animal well-being and the productivity. A wide range of abiotic stressors has been identified, such as social interactions or rough handling, common farm practices (e.g. castration, dehorning, teeth clipping, shoeing, weaning crowding etc), improper feeding, exposure to adverse climatic conditions, exercise, work and transport. Any imbalance in these factors will first induce animal adaptation and tolerance, which may result in behavioral, biological, and physical responses. In case non-adapted conditions are not rapidly corrected, the tolerance threshold may be exceeded and the animal will externalize the imbalance via stress. Stress is a reflex reaction revealed by the inability of an animal to cope with its environment, which may lead to many unfavorable consequences, ranging from discomfort to death. Stress-triggering stimuli are not necessarily painful but may activate physiological responses and the animal could develop behavioral, autonomic, endocrine or immune response to maintain homeostasis. In case the animal is unable to withstand stress, the consequences will be abnormal biological functions, which could lead to the development of psychosomatic disease, immunosuppression, reduced efficiency of production and reproduction. Stress affects ability to perform and may make animals more susceptible to physio-pathological disorders. All these detrimental animal responses are especially in relation, at least partly, to impaired gut physiological function.
The barrier formed by the intestinal epithelium separates the external environment (i.e. the contents of the intestinal lumen) from the body. The intestinal epithelium is composed of a single layer of epithelial cells and serves two crucial functions, which may seem conflicting. On one hand, it must act as a barrier to prevent the entry of microorganisms that inhabit the gastrointestinal tract, as well as undesirable components that may be present in the intestinal chyme. On the other hand, it must facilitate the uptake of dietary nutrients, electrolytes, water and various other beneficial substances from the intestinal lumen.
The gut epithelium maintains its selective barrier function through the formation of complex protein-protein networks that mechanically link adjacent cells and seal the intercellular space, especially through the involvement of tight junctions. Each stress response of the animal will challenge the integrity of the mucosal barrier and the intestinal epithelium will need to adapt to a multitude of signals in order to perform the complex process of maintenance and restitution of its barrier function. A well-functioning epithelium is also crucial to optimize the absorption of dietary nutrients that are essential for efficient metabolic processes. Conditions able to help the animal to maintain its gut barrier integrity are then the touchstone for steady physiological status required to face adverse rearing conditions.
To overcome the impact of stressful conditions on gut physiological function and animal productivity, many different strategies have been proposed. The current solutions are generally prophylactic with the use of dietary antibiotic-growth promoters (AGP) and biosecurity measures to control environmental parameters. With the concerns regarding antibiotic resistance and the difficulties related to the identification of proper management practices/biosecurity measures, and their interactions, alternative prophylactic methods have been developed in order to provide complementary solutions to an integrative approach at the farm level. Combination of feed additives to support host digestive processes and gut physiological function are especially the focus of many research teams around the world. These include probiotics, prebiotics, short- and medium-chain fatty acids, herbal compounds, among other molecules (Van Immerseel et al. (2017), Microb. Biotechnol., 10(5): 1008-1011). As a key issue in production animals is digestibility of nutrients and energy harvest from the diet, supplementation with digestive enhancers (such as enzymes) is also used to manage dietary stresses. However, in addition to complicating diet formulation and associated costs, the interactions induced by supplementing different feed additives are not always fully described and well-known.
Therefore, it would be desirable to develop functional ingredients allowing both to bring essential nutrients such as protein and amino acids, and to offer protection against multifactorial stress, while maintaining gut barrier integrity and preventing the transfer of undesirable compounds into the body, in order to reduce diet and veterinary costs, while securing rearing practices.
Thraustochytrid microalgae are known for their use in biofuel production and as a source of polyunsaturated fatty acids. It has also been shown, in WO2017/012931, that protein-rich biomass of Thraustochytrids can improve animal performance in animals receiving a standard starter diet based on corn and soybean meal (which is optimal for chicken metabolism), and which are not submitted to stressful conditions (such as nutritional/dietary stressor(s)). WO2004/080196 discloses animal feed comprising lower fungal biomass (e.g. from Thraustochytrid microalgae), which can have a wide range of effects, including the improvement of gut function, stimulation of probiont colonization, and improved food conversion. Similarly here, the animals are not submitted to stressful conditions (whether in terms of environmental conditions e.g. stocking density, or diet).
The invention disclosed in US 2017/0369681 consists in a combination of microalgae (including Thraustochytrid microalgae) and soluble indigestible fibers, having a synergistic effect on the stimulation of bacteria of the intestinal flora, their enzymes production, as well as the protection of intestinal health through the release of active agents from the lysed microalgae (whereas such effects are not observed with the microalgae alone). Moreover, it was disclosed in this document that microalgae (and more particularly, Chlorella vulgaris, Chlorella saccharophila, Scenedesmus, Chlamydomonas reinhardtii or Dunaliella salina), can adsorb toxins synthesized by enteropathogenic bacteria, on their cell wall (some of these toxins being implicated in numerous intestinal diseases, including inflammatory diseases). However, it is known that cell wall composition can vary significantly between different microalgae, and in particular, the cell wall composition of Thraustochytrids is very different from the cell wall composition of Chlorella (Domozych et al (2012), Frontiers in Plant Science, 3:82; Gerken et al (2013), Planta, 237(1):239-253; Darley et al (1973), Arch. Mikrobiol., 90:89-106). In Bedirli et al (2009), Clinical Nutrition 28: 674-678, it was also shown that different microalgae can have different effects; in particular, Chlorella microalgae, but not Spirulina microalgae, could reduce intestinal translocation of bacteria and endotoxin in obstructive jaundice.
It has now been discovered by the inventors, completely unexpectedly, that Thraustochytrid microalgae allow both to bring essential nutrients such as protein and amino acids, and to offer protection against multifactorial stress, while maintaining gut barrier integrity and preventing the transfer of undesirable compounds into the body.
Therefore, the present invention relates to the use of a Thraustochytrid biomass for maintaining gut barrier function in an individual.
In the context of the present invention:
Preferably, the Thraustochytrid biomass is used for maintaining gut barrier function in an individual, preferably an animal, who is submitted to stressful or challenging conditions, in particular stressor(s) or challenge(s) which can impair gut physiological function. In animal production, a wide range of abiotic stressors has been identified, which can in particular be related to:
Stressors can in particular occur in intensive animal breeding/livestock operations and adverse rearing conditions.
Preferably, the Thraustochytrid used according to the present invention is selected from the group consisting of:
Still preferably, the Thraustochytrid used according to the present invention is of a genus selected from the group consisting of the genera Aurantiochytrium and Schizochytrium; more preferably of a species selected from the group consisting of the species Aurantiochytrium mangrovei and Schizochytrium sp.; even more preferably of a strain selected from the group consisting of the strains Aurantiochytrium mangrovei CCAP 4062/2 deposited 20 May 2014 at CCAP (CULTURE COLLECTION OF ALGAE AND PROTOZOA, SAMS Research Services Ltd., Scottish Marine Institute, OBAN, Argyl PA37 1QA United Kingdom), Aurantiochytrium mangrovei CCAP 4062/3 deposited 20 May 2014 at CCAP, Aurantiochytrium mangrovei CCAP 4062/4 deposited 20 May 2014 at CCAP, Aurantiochytrium mangrovei CCAP 4062/5 deposited 20 May 2014 at CCAP, Aurantiochytrium mangrovei CCAP 4062/6 deposited 20 May 2014 at CCAP, Aurantiochytrium CCAP 4062/1 deposited 21 Jun. 2013 at CCAP, Schizochytrium sp. CCAP 4087/3 deposited 20 May 2014 at CCAP, Schizochytrium sp. CCAP 4087/1 deposited 28 Feb. 2012 at CCAP, Schizochytrium sp. CCAP 4087/4 deposited 20 May 2014 at CCAP and Schizochytrium sp. CCAP 4087/5 deposited 20 May 2014 at CCAP.
In a preferred embodiment, the Thraustochytrid used according to the present invention is Aurantiochytrium mangrovei FCC1325 (accession number CCAP 4062/5).
The Thraustochytrid biomass used according to the present invention may be used in different forms. It can for instance be in the form of fresh biomass (which can be separated from the culture medium by centrifugation, filtration, decantation and/or any other technique well-known to the skilled person), or it may have undergone some modifications; for instance it may have been submitted to lysis, transformation by fermentation and/or drying. In particular, drying can be performed by any technique well-known to the skilled person, such as spray-drying, lyophilization, fluidized bed, high vacuum evaporation or fluid bed granulation.
The Thraustochytrid biomass used according to the present invention may be used directly as a dietary supplement, or added to or incorporated into a compound feed/balanced diet, a food product or a food composition. In these latter cases, the Thraustochytrid biomass used according to the present invention may be mixed with any other additive, carrier or support, used in the field of food or feed, for human or animal consumption, such as for example food preservatives, dyes, flavor enhancers or pH regulators.
Preferably, the Thraustochytrid biomass used according to the present invention is a feed ingredient (i.e. intended to be incorporated into a compound feed, at an inclusion level ranging from 1% to 60% (w/w), preferably ranging from 1% to 20% (w/w), more preferably ranging from 3% to 8% (w/w)), a feed additive (i.e. intended to be incorporated into a compound feed, at an inclusion level inferior to 1% (w/w)), or is comprised in a compound feed, a food product or a food composition.
The Thraustochytrid biomass used according to the present invention may be intended for animal or human nutrition. Preferably, it is intended for animal nutrition, still preferably for livestock animals or leisure animals feeding. More preferably, it is intended for livestock animals feeding (especially in particularly intensive livestock operations).
These feeds typically appear in the form of flours, crumbles, pellets or slop, into which the Thraustochytrid biomass used according to the present invention can be incorporated. For intensive animal breeding operations, the feeds may comprise, in addition to the Thraustochytrid biomass, a nutritional base and nutritional additives. The essential part of the animal's feed ration thus generally consists of the “nutritional base” and the Thraustochytrid biomass. This base may consist, by way of example, of a mixture of cereals, proteins and fats of animal and/or plant origin. Nutritional bases for animals are adapted to the feeding of these animals and are well-known to the skilled person. In the context of the present invention, these nutritional bases may comprise, for example, corn, wheat, pea and soybean. These nutritional bases are adapted to the needs of the various animal species for which they are intended. These nutritional bases may already contain nutritional additives such as vitamins, mineral salts and amino acids. The additives used in animal feed may be added to improve certain characteristics of the feeds, for example to enhance the flavor thereof, to make the raw materials of the feeds more digestible for the animals or to protect the animals. They are frequently used in large-scale intensive breeding operations. The additives used in animal feeds can be divided into: technological additives (e.g. preservatives, antioxidants, emulsifiers, stabilizers, acidity regulators and silage additives), sensory additives (e.g. flavors, dyes), nutritional additives (e.g. vitamins, amino acids and trace elements), zootechnical additives (e.g. digestibility enhancers, intestinal flora stabilizers), coccidiostats and histomonostats (pesticides).
Even more preferably, the Thraustochytrid biomass used according to the present invention is intended for livestock animals feeding, wherein livestock animals are selected from the group consisting of cattle, sheep, pigs, rabbits, poultry and horses.
All the above-mentioned preferential features of the invention can be considered separately or in any combination.
Another object of the present invention concerns a process for maintaining gut barrier function in an individual, comprising a step of administering to said individual a Thraustochytrid biomass as described previously, and having preferably any of the above-mentioned preferential features, considered separately or in any combination.
Top: Length of colon (in cm/kg of body weight (BW)) of 16-day old chickens. ** P<0.05. Bottom: Visual aspect of colon mucosa. A: Control group receiving the basal diet without DSS administration. B: Control group receiving the basal diet with DSS administration. C: Experimental group receiving the diet containing 5% of Aurantiochytrium mangrovei with DSS administration.
The present invention is illustrated non-exhaustively by the following examples. These examples are intended for the purpose of illustration only and are not intended to limit the scope of the present invention.
Material and Methods:
Caco-2 cells were used as a model of intestinal epithelial cells. Cells were routinely grown in culture media (DMEM) supplemented with 10% fetal calf serum and 1% antibiotics (streptomycin penicillin solution). Cells were grown in 75 cm2 ventilated flasks maintained at 37° C. in a 5% CO2 incubator. Cells were routinely passaged using trypsin-EDTA solution. For the assay, cells were seeded onto 12-well inserts (Thincert, Greiner, pore size 0.4 μm) at an initial density of 200,000 cells/cm2 and let to differentiate for 10-14 days post-seeding before being used, with medium changes every two days. Cell differentiation was confirmed by reading the TER, using a Volt/Ohm meter (Millipore), at the beginning of the experiment, when the TER value reached 600 Ohm/cm2.
Deoxynivalenol (DON) was used to induce an increased permeability, and various forms of microalgae (Aurantiochytrium mangrovei FCC1325) preparations were tested for their ability to reduce the effect of DON:
Both lyophilized (ML) and digested lyophilized (MLD) microalgae powders were resuspended initially at 0.8 mg/ml in buffer (fresh culture medium of the microalgae).
After differentiation, Caco-2 cells were put in contact, during 48 or 72 h, with or without DON at different concentrations (0, 6.25, 12.5, 25, 50 or 100 μM), and with activated charcoal at 1% (w/v) as positive control, or with or without one of the microalgae preparation type at different concentrations (1, 5, or 20% v:v, final dilution), each added on the apical side. At the end of the incubation time, the TER was measured using a Volt/Ohm meter (Millipore), and results were expressed in percentages of the control put in contact with the same concentration of DON but not with the tested product (microalgae or charcoal). Each condition was tested in triplicates (n=3).
Results:
The addition of DON only to the cell medium induced a reduction of the TER (corresponding to an increased permeability), which was even more pronounced as the concentration of DON increased (see “control” condition in
The half-maximal inhibitory concentration (IC50) is a measure of the potency of a substance in inhibiting a specific biological or biochemical function. This quantitative measure, typically expressed as molar concentration, indicates how much of a particular substance (inhibitor) is needed to inhibit a given biological process by half. The analysis of IC50 at 48 h incubation (Table 1) clearly confirmed the ability of the microalgae to prevent the DON effect on the Caco-2 TER. At 1 and 5%, the fresh and lyophilized microalgae biomass appeared to be the most protective (IC50 values 3-10 times higher, compared to control), while at a concentration of 20%, the digested lyophilized microalgae showed better protection than the fresh and lyophilized biomasses.
After a 72-h incubation, some of the microalgae showed higher preventive effect than charcoal, and the most efficient prevention was obtained with microalgae at 20% (
Material and Methods:
In order to test the ability of microalgae to reduce/prevent the effect of DON on nutrient absorption through epithelial cells, Caco-2 cells were exposed to a metabolically active dose of DON, in the absence or presence of Aurantiochytrium mangrovei FCC1325 microalgae (lyophilized microalgae “ML”, or digested lyophilized microalgae “MLD”), at a dose of 1% or 5%. Two main types of nutrients were considered (i.e. glucose and amino acids—more particularly Methionine, Lysine and Threonine), and the following measurements were carried out:
Briefly, Caco-2 cells were cultured and seeded onto 12-well inserts, as described in Example 1, and then let to differentiate for 16-21 days post-seeding before being used, with medium changes every two days. When differentiated, Caco-2 cells were incubated or not with DON at 10 μM (apically added), in the absence or presence of 1 or 5% (v:v final dilution, apically added) of microalgae preparation (ML or MLD). Both ML and MLD powders were resuspended initially at 0.8 mg/ml in buffer (fresh culture medium of the microalgae). Caco-2 cells were incubated for 12, 24 or 48 hours before nutrient uptake was measured.
At the end of the incubation period, inserts were washed twice with PBS++. Inserts were then washed twice with uptake buffer (Ringer Hepes buffer) with or without sodium. Uptake buffer composition was:
After an equilibration period of 15 min at 37° C., uptake assay was initiated by the addition of D-Glc, L-Lysine, L-Methionine or L-Threonine diluted in the appropriate Ringer Hepes buffer (400 μl) and added apically onto Caco-2 inserts (final concentration of 100 μM of D-Glc and 400 μM for amino-acids), the basolateral compartment being filled with 400 μl of buffer. Inserts were kept incubated at 37° C. during the uptake assay. After 15 minutes incubation, 30 μl of media were collected from apical or basolateral compartments and stored at −20° C. until nutrient quantification. Residual concentrations of D-Glc or L-amino acid present in the apical compartments were measured using enzyme-based quantification assay kits (Glucose Colorimetric/Fluorometric Assay Kit, Sigma-Aldrich).
Uptakes were expressed as:
Results:
D-Glc Uptake after Exposure to DON, in the Absence or Presence of Microalgae
12 h Incubation
At 12 h incubation (see Table 2), ML and MLD suppressed DON effect on total Glc uptake (ML 5% and MLD 1/5%), passive uptake (ML 5% and MLD 1/5%) and SGLT-1 activity (all ML and MLD).
24 Incubation
At 24 h incubation, ML and MLD reversed/prevented DON-mediated inhibition of total, passive and active D-Glc uptake (see Table 3).
48 h Incubation
At 48 h incubation, similarly as for the 24 h incubation, ML and MLD reversed/prevented DON-mediated inhibition of total, passive and active D-Glc uptake.
L-Amino Acids Uptake after Exposure to DON, in the Absence or Presence of Microalgae
12 h Incubation
ML and MLD did not prevent the effect of DON on total or passive L-Lys absorption but ML 1% and MLD 1% were able to prevent L-Lys active uptake inhibition by DON (Table 5).
Contrarily to L-Lys and L-Thr that were inhibited by DON, L-Met active uptake was stimulated by DON. ML and MLD were able to limit L-Met uptake stimulation by DON (Table 6).
ML but not MLD were able to limit L-Thr active uptake inhibition by DON (Table 7).
24 h Incubation
Table 8 shows that ML 1% (but not the other forms of microalgae) was able to reverse the effect of DON on active L-Lys uptake.
Table 9 shows that both ML and MLD prevented DON effects on L-Met active transport.
Table 10 shows that ML and MLD 5% prevented the inhibition of L-Thr active uptake by DON.
48 h Incubation
At 48 h incubation, DON stimulated active L-Lys uptake. This effect was prevented by ML but not by MLD (Table 11). At 48 h incubation, DON stimulated active L-Met uptake. This effect was prevented by ML and MLD 5% but not by MLD 1% (Table 12).
Table 13 shows that both ML and MLD prevented partially the inhibition of active L-Thr uptake by DON.
The most important uptakes to be considered are total uptake (in order to have a global view of nutrient uptake capacity) and active uptake (in order to evaluate anti-diarrheal nutrient uptake activity). Overall, results are consistent with previously published results obtained with radioactive nutrients and HT-29-D4 cells, confirming that DON at 10 μM alters intestinal nutrient uptake. These first observations suggested that the lyophilized microalgae with or without pre-digestion is able to partially reverse/prevent DON-mediated impact on total, passive, and active uptake of glucose, Lysine, Threonine, and Methionine.
Material and Methods:
Results:
| Number | Date | Country | Kind |
|---|---|---|---|
| PCT/EP2019/052527 | Feb 2019 | EP | regional |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2020/052474 | 1/31/2020 | WO | 00 |
