In the present study, two experimental isoproteic, isolipidic and isoenergetic microdiets were formulated and elaborated at Laboratorio Nacional de Nutrigenómica y Microbiómica Digestiva Animal (LANMDA), Universidad Michoacana de San Nicolás de Hidalgo, Mexico, according to Martínez-Ángeles et al. (2022). The first microdiet was supplemented with Lactobacillus acidophilus La-14 (FloraFit, Dupont, Mexico), and the second was used as a control diet (without L. acidophilus supplementation). Both microdiets were formulated to contain 520 g Kg-1 protein and 220 g Kg-1 lipids (Table 1). Proximal analyzes of the ingredients and microdiets were performed according to the following AOAC (2000) methods and equipment: moisture (Fisher Scientific Isotemp oven), crude protein (Leco FP-528, Dumas method; Ebeling 1968), crude lipid (ether extract; Soxtec Avanti 2050), and crude ash (Fisher Scientific muffle furnace). All samples were analyzed in triplicate (Table 1).
Table 1
Dietary formulation g Kg− 1 (protein / lipid level 520 / 220 g Kg− 1) and chemical composition (mean ± standard deviation) of the evaluated microdiets.
| Experimental Diets |
| Control | Lactobacillus acidophilus La-14 |
Ingredients (g Kg− 1) | | |
Protein Ingredientsa | 673.8 | 673.8 |
Cod liver oil | 97.9 | 97.9 |
Canola oil | 90.7 | 90.7 |
Soy lecithin | 19.3 | 19.3 |
Corn starch | 31.4 | 31.4 |
Guar gum | 20.0 | 20.0 |
Mineral premixb | 15.0 | 15.0 |
Vitamin Cc | 3.4 | 3.4 |
Choline chlorided | 3.0 | 3.0 |
Vitamin premixe | 15.0 | 15.0 |
Othersf | 30.5 | 30.5 |
Lactobacillus acidophilus (CFU mL− 1) | 0.0 | 2.98 x 109 |
Chemical Composition* (g Kg− 1) (n = 3) | | |
Crude Protein | 539.3 ± 1.2 | 543.1 ± 2.3 |
Crude Lipids | 223.5 ± 1.5 | 224.2 ± 2.2 |
Ash | 58.2 ± 1.1 | 57.6 ± 0.5 |
Moisture | 29.9 ± 0.8 | 30.7 ± 1.2 |
a Fresh California squid, fresh grouper fillets, dry krill (Tetra), egg albumin (Abaquim S.A.), Whey Protein Concentrate (WPC80; América alimentos), calcium caseinate (Habacuq S.A. de C.V.), wheat germ.
b Mineral premix: macro elements and trace elements (DSM Nutritional products).
c L-Ascorbyl-2-Poliphosphate (AsPP), Rovimix®Stay C®35 (DSM Nutritional products).
d Choline Chloride (DSM Nutritional products).
e Vitamin premix (DSM Nutritional products).
f Butyl hydroxytoluene (BHT: antioxidant), Crystaline Taurine, Betaine, orozuz powder, apple extract.
* Chemical composition of the commercial microdiet was analyzed in LAMNDA, Universidad Michoacana de San n.
Nicolás de Hidalgo.
Micro-encapsulated diets were prepared by spray drying (Niro Atomizer, Copenhagen, Denmark, MOBILE MINOR™ 2014, MM-PSR model) with an inlet temperature of 185°C, an outlet temperature of 75°C, and a feeding rate of 30 mL min-1, as previously described in detail in Martínez-Ángeles et al. (2022).
Before microdiet elaboration, the bacterial strain L. acidophilus was cultured in MRS Agar Lactobacilli media (Difco TM) at 37°C for 24 hours. Subsequently, a sample of 1 mL was taken from the culture medium, diluted in saline water (1:3), and 0.1 mL of the dilution was placed inside a Neubauer chamber to count the number of bacteria per millilitre (CFU mL-1), using the following equation:
CFU mL-1= ((Counted bacteria)/(Counted area mm2 * Chamber depth mm))* dilution
The initial concentration of L. acidophilus (2.98 x 109 CFU mL-1) was directly added to the homogeneous liquid mixture of the supplemented microdiet ingredients before spray drying. After preparation, both microdiets were packed in hermetic plastic bags and stored at 4°C. Probiotic viability was evaluated in the L. acidophilus supplemented diet after 24 h of the spray drying and every 15 days during six months of storage. The same procedure was performed in the control microdiet to evaluate the presence of lactic acid bacteria (LAB) during the trial to ensure no cross-contamination.
Two types of agar culture media specific for LAB (17.5 g MRS agar, Difco TM) were elaborated, one dissolved in 250 mL of ultra-filtered marine water (35 g L-1) and the other in 250 mL of distilled water. A 100 mL dilution (10− 3) with 0.1 g of each microdiet (L. acidophilus and control) was carefully spread in Petri dishes in each culture media and incubated at 37°C for 24 hours. Finally, bacterial colonies (CFU g-1) were counted on a dark field colony counter and calculated with the following equation:
CFU g-1= (Number of colonies*Dilution factor)/Sample weight g
The results obtained in these tests were normally distributed and homoscedastic and were analyzed using one-way ANOVA, with a significance level of α = 0.05. A Tukey posthoc test (α = 0.05) was performed using SigmaPlot statistical software (ver. 14.5).