Production of ingredients
An exhaustive bibliographic review process, both external and own, was carried out to verify the theoretical efficacy of fungi and microalgae-based products applied to poultry. This point also has a direct impact on the formulation of the nuclei and their dosage, since the most suitable species of fungi and microalgae have been determined, as well as their format and dosage.
Once those species were selected that, according to bibliography, proved to have a greater antimicrobial, immunomodulatory and/or anti-inflammatory potential, pilot scale production of the fungi and microalgae was started in order to generate sufficient quantities of these ingredients to begin in vitro tests. The process of generating the compounds of interest and preparing them for laboratory test was also optimized.
Tubular photobioreactor with a C. vulgaris culture at low light intensity.
The scaling-up process began once the first preliminary results were obtained. These showed that the microalgae with the greatest potential was Chlorella vulgaris (Cv) and two samples of fungi, made from the nuclei of “oyster mushroom” (P. ostreatus), “reishi” (G. lucidum) and “shiitake” (L. edodes). Therefore, the production of these species was scaled up, increasing efficiency in a process of continuous improvement, taking into account proper preservation, maintenance and traceability.
In vitro screening of fungi and microalgae
The first in vitro tests of the ingredients and their combination were carried out with a total of 5 samples of 90g of microalgae and 5 samples of 100g of fungi.
First, a selection of the strains of interest to act as targets in the different assays was carried out. The main pathogens of interest in poultry farming can be classified into several groups: bacteriophages, viruses, fungi and others (mainly parasites). Taking into account the most relevant ones in the field or with a greater relationship with zoonosis, E. coli, Campylobacter and Salmonella were selected as pathogens of interest, coming from our own biological and genetic resources as well as from field samples.
To determine their antimicrobial capacity, in vitro tests were carried out using culture media to diagnose the degree of inhibition that the extracts exert on the target microorganisms and, if necessary, to determine the Minimum Inhibitory Concentration (CMI). Two analytical techniques were used: the ecometric method and the turbidity method.
No antimicrobial activity was detected in any of the ingredients evaluated.
Immunomodulatory and anti-inflammatory effect
Macrophages are the cells of the immune system that orchestrate the innate and adaptive response to a perturbation, being the most evaluated in in vitro assays. On the other hand, cytokines are the humoral factors that produce the immune response, acting primarily as regulators of the immune and inflammatory responses. In the case of the MICOALGA-FEED assays, these were performed on the avian macrophage cell line HD11, with the cytokines of interest being the following: IL-1β, IL-6 and IL-10.
On the other hand, poultry are constantly exposed to a large number of microorganisms, some of them pathogenic and others typical of the flora. Therefore, the defense cells that come into contact with the MICOALGA-FEDD feed will be, depending on the state of poultry at the moment, active or inactive. For this, is essential to evaluate the dual effect.
When macrophages are inactive, the immunostimulatory effect is evaluated: an increase in the concentration of cytokines IL-1β and IL-6 after the addition of the ingredients means an immunostimulatory effect, since it prepares macrophages against threats.
On the contrary, when the macrophage is active, the anti-inflammatory effect is evaluated: a decrease in the concentration of cytokines, implies an anti-inflammatory effect, since it reduces inflammation.
However, before evaluating the functional effect of the products, it is necessary to know their biocompatibility with the HD11 cell line. After performing this analysis, it was observed that the maximum biocompatible concentration of microalgae was 250 ug/mL. on the other hand, the maximum biocompatible concentration of fungi is 500 ug/mL. in order to obtain comparable results between fungi and microalgae, the evaluation of the immunomodulatory and anti-inflammatory effect was performed with a concentration of 250 ug/mL.
As mentioned above, to assess the immunomodulatory effect of the ingredients, HD11 cells (avian macrophages) are “inactive” and treated with the samples of interest.
In the case of fungi, all the samples provided have shown to possess an immunomodulatory effect, by increasing in a statistically significant way IL-1β and IL-6 cytokines.
As for microalgae, all but 1 have demonstrated such an immunostimulatory effect.
As for the anti-inflammatory capacity, contrary to the previous case, HD11 cells (avian macrophages) are exposed to an activator that increases the expression of proinflammatory cytokines in these cells and simulates an inflammatory context. Fungal and microalgae samples are then incorporated to evaluate the decrease of these cytokines, leading to a reduction of inflammation and thus and anti-inflammatory effect of the samples.
An anti-inflammatory effect has been detected in samples M1 and M4 of the fungi and in sample Cv of the microalgae.
To summarize, as shown in the tables on the right, all fungal samples have shown an immunostimulatory effect on inactive defense cells. In addition, samples M1 and M4 show an anti-inflammatory effect when defense cells are activated. Therefore, M1 and M4 are the samples of greatest interest because of their dual capacity.
Table 1. Summary of the immunomodulatory and anti-inflammatory effect, respectively, of the funal samples.
Table 2. Summary of immunomodulatory and anti-inflammatory effect, respectively, of the microalgae samples.
The microalgae samples have an immunostimulatory effect on inactive defense cells. When the defense cells are activated, only the Cv sample (at a concentration of 50 ug/mL) demonstrated an anti-inflammatory effect. Therefore, the Cv sample is the one of greatest interest.
Based on these results, it was decided to evaluate the immunomodulatory and anti-inflammatory capacity of the combination of the fungi M1 and the microalgae Cv, and of the combination of the fungi M4 with Cv. Finally, the most satisfactory results were for the first combination (M1 + Cv). Likewise, these in vitro studies showed that 5 times more M1 fungi ingredients should be included than Cv microalgae to have an effective combination in the 2 scenarios evaluated.
Feed production and evaluation of functional effects
Feeds were developed to which the microalgae sample Cv, the fungi sample M1 and a combination of both were incorporated, in addition to the control feed. These 4 feeds were duplicated to incorporate two different salt correctors, making a total of 8 MICOALGA-FEED feeds, as shown below:
Following the same protocol as for the ingredients, an analysis of the antimicrobial, immunomodulatory and anti-inflammatory capacity of the 8 MICOALGA-FEED feeds was performed. The table on the left refers to the results of the feeds that included salt corrector 1, and the table on the right refers to the results of the feeds that included salt corrector 2. In terms of antimicrobial capacity, no feed gave positive results.
Simulation of poultry gastrointestinal digestion
The capacity of the feed is as important as that of the digestates obtained after the passage of the feed through the avian gastrointestinal system, since it is the latter that are assimilated by the animal. The feed digests were obtained with a simulator of avian gastrointestinal digestion that includes the crop, stomach and small intestine, which reproduces the conditions of temperature, time and pH of each section of the digestive tract.
None of the digests demonstrated antimicrobial capacity. As for the immunomodulatory and anti-inflammatory capacity, the results obtained are shown in the following table.
Conclusions of the in vitro tests
Antimicrobial capacity. The results under the conditions tested have not been satisfactory in the case of the ingredients and their combinations. Neither has it been possible to find antimicrobial activity in the formulated feeds nor in their digests.
Immunomodulatory effect. The one that has shown the best in vitro results is the one that incorporates the combination of fungi (M1) and microalgae (Cv), since it shows an immunostimulant effect both in the feed and in the digests, independently of the salt corrector.
Anti-inflammatory effect. None of the formulated feed have shown a negative result against immunomodulatory or anti-inflammatory activity in both feeds and their digests.
Conclusions of the in vivo tests
During the last phase of the project, an in-vivo trial in an experimental farm was carried out, in which different parameters were measured on the farm and in the poultry processing plant.
At the farm, the weekly consumption and weights per flock, the daily mortality record, total water consumption, temperature and relative humidity, the study of animal welfare indicators at 15 and 42 days, and finally, fecal samples were taken at 15, 28 and 42 days. As for the poultry processing plant, the homogeneity of the flock and the percentage of seconds, yields, a study of meat quality in terms of pH, color and myopathies were measured, blood samples were taken for a hemogram and the determination of cytokines and, in addition, a study of shelf life and sensory analysis was made on meat from different batches.
It was concluded, in relation to the results of zootechnical indexes, that there were very significant differences with the MICOALGA-FEED feeds with respect to the commercial ones. The main differences were observed in weight and consumption, while in the feed conversion rate, the expected values were recovered.
Finally, it was observed that mortality in those birds fed with the MICOALGA-FEED feed decreased significantly.