In vitro acid digestibility of four marine species using the digestive gland of adult Octopus vulgaris.
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1
Instituto Español de Oceanografia - Centro Oceanografico de Vigo, Spain
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2
Unidad Multidisciplinaria de Docencia e Investigación, Facultad de Ciencias – UNAM, Mexico
Abstract
The determination of in vitro digestibility of acid proteases in the digestive gland (DG) of adult O. vulgaris (>1,2 Kg) was performed, using four different preys (Mackrel, Scomber scombrus; Green crab, Carcinus maenas; Swimming crab, Necora puber; Shortfin squid, Illex coindetti). For each prey, four treatments were applied: 1) Frozen, 2) boiled at 100ºC for 1 minute, 3) Lyophilized and 4) Oven dried at 70ºC. A pH-Stat was used to determine in vitro digestibility at pH 3. Results showed that digestibility was higher (p<0.05) for frozen and lyophilized diets (and not different between these two treatments in all four diets tested; p>0.05), compared to the other two treatments that suffered heating (boiled and oven dried), regardless of the diets tested. Regarding acid proteases metabolism, which is the most important for O. vulgaris, results indicate that heat treatments close and/or higher than 70ºC promote changes in the different diets that prevent their assimilation by the DG of O. vulgaris.
Material and methods
The DG of 30 adult octopuses were removed shortly after capture and frozen. From these, 8 whole DG were grinded together and the resulting paste was placed in 1,5 ml tubes and centrifuged at 12.000 rpm for 30 minutes at 4ºC. The supernatant was then collected and placed in other tubes (800 µl each) and stored at -80ºC. Acidic protease activity of O. vulgaris DG at pH 3 was evaluated according to Anson (1938) modified, using a solution of 0.5% (w/v) bovine hemoglobin (Sigma H2625) in 100 mM universal buffer (Stauffer, 1989). Total acid protease activity for the DG was 107026 IU mg protein-1. Trials were run with the pH-Stat, with 10 replicates for each diet and treatment: 1) Frozen, 2) boiled at 100ºC for 1 minute, 3) Lyophilized and 4) Oven dried at 70ºC. Also, to eliminate activity from the tissue itself, for each diet and treatment, 10 runs with enzyme and 10 runs without enzyme were performed, and the difference in activity was used to determine residual activity of each diet and treatment.
Degree of protein hydrolysis (DH)
The DH of the diets was assessed in vitro using the pH-stat system with O. vulgaris DG (Lemos, 2004; Lemos et al., 2004). The DH was determined for each diet and their 4 treatments. Samples were pre-stirred with 50 ml of distilled water in the hydrolysis vessel for until the pH of the suspension was adjusted to near 3.0 (with HCl at 0.1 N). The stirring preparation period is important to stabilize the pH of the suspension to avoid non-enzymatic pH shifts during hydrolysis. The DH was determined with a pH-stat titration method (Pedersen and Eggum, 1983) using 902 Tritando© (Metrohm, Switzerland) with Tiamo 2.3© software. The DH determination principle is based on the acidification of the reaction enzyme-substrate by the release of H+ after enzymatic hydrolysis of peptide bonds.
The DH for a 15-min reaction was calculated as (Adler-Nissen, 1986; Lemos and Motikawa, 2006): DH (%) = (B × Nb × 1/ α × 1/Mp × 1= Htot) × 100
where B=mL of standard acid (0.1 N HCl) consumed to maintain the reaction mixture at pH 3.0; Nb=normality of the titrant; 1/α=pK for amino groups at the given temperature, pH3.0 (1.5 and 1.4 for 25 and 30 °C, respectively); Mp=crude protein mass in sample (g); Htot=total number of peptide bonds in the protein substrate (7.8–8.6 meqv g protein−1, depending on ingredient protein nature, Adler-Nissen,1986). The pH-stat reaction temperature tested was 20 °C, maintained using a jacketed reaction vessel connected to a circulated bath.
Each of the four diets and their treatments were stored at -80ºC, and defrosted prior to their use in each trial.
Results and discussion
The “in vitro” digestibility was higher (p<0.05) for the frozen and lyophilized diets (than the other two treatments) (Table 1). Regarding acid proteases metabolism, which is the most important for O. vulgaris, results indicate that heat treatments close and/or higher than 70ºC promote changes in the different diets that prevent their assimilation by the DG of O. vulgaris.
Previous studies demonstrated that O. maya adults fed with prepared diets based on commercial fish flowers was negative (Domingues et al., 2007). Similarly, boiled shrimp fed to cuttlefish (Sepia officinalis) juveniles promoted negative growth, compared to thawed and lyophilized shrimp, which promoted good growth, with no differences between the two treatments (Domingues et al., 2009). Results obtained in the present study demonstrate that, besides the digestive problems that provokes cooked diets in absorption of nutrients (Rosas et al. 2013), heat reduces the protein hydrolysis by enzymes, due to the effects that hot produce in protein that makes impossible to digest by several octopus species (Santé-Lhoutellier et al. 2008)
Table 1 – Degree of hydrolysis (DH, %) of 4 different species and four treatments each, using the digestive gland of adult O. vulgaris. Letters indicate significant differences (p<0.05).
References
Domingues, P., Marquez, L., López, N., Rosas, C. (2009) Effects of food thermal treatment on growth, absorption, and assimilation efficiency of juvenile cuttlefish, Sepia officinalis. Aquaculture International 17: 283-299.
Domingues, P., López, N., Muñoz, J.A., Maldonado, T., Gaxiola, G., Rosas, C. (2007) Effects of a dry pelleted diet on growth and survival of the Yucatan octopus, Octopus maya. Aquaculture Nutrition 13: 273-280.
Rosas C, Valero A, Caamal-Monsreal C, Uriarte I, Farias A, Gallardo P, Sánchez A, Domingues P (2013) Effects of dietary protein sources on growth, survival and digestive capacity of Octopus maya juveniles (Mollusca: Cephalopoda). Aquaculture Research 44: 1029–1044
Santé-Lhoutellier V, Astruct T, Marinova P, Greve E, Gatellier P (2008) Effect of meat cooking on physicochemical state and in vitro digestibility of myofibrillar proteins. JAgricFoodChem 56: 1488-1494
Keywords:
octopus vulgaris,
DIETS,
in vitro acid digestibility,
digestive gland,
Heating
Conference:
XIX Iberian Symposium on Marine Biology Studies, Porto, Portugal, 5 Sep - 9 Sep, 2016.
Presentation Type:
Poster Presentation
Topic:
4. FISHERIES, AQUACULTURE AND BIOTECHNOLOGY
Citation:
Domingues
PM,
Gallardo
P,
Sanches
A,
Chimal
ME and
Rosas
C
(2016). In vitro acid digestibility of four marine species using the digestive gland of adult Octopus vulgaris..
Front. Mar. Sci.
Conference Abstract:
XIX Iberian Symposium on Marine Biology Studies.
doi: 10.3389/conf.FMARS.2016.05.00209
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Received:
27 Apr 2016;
Published Online:
03 Sep 2016.
*
Correspondence:
MD, PhD. Pedro M Domingues, Instituto Español de Oceanografia - Centro Oceanografico de Vigo, VIGO, 36390, Spain, pedro.domingues@vi.ieo.es