EP4213639A1

EP4213639A1 - New hydrolysates to control infections, methods and uses thereof

Info

Publication number: EP4213639A1
Application number: EP21810714.2A
Authority: EP
Inventors: Maria Manuela ESTEVES PINTADO; Helena Maria ARAÚJO RODRIGUES; Miguel Fernando RIBEIRO PEREIRA; Benjamin COSTAS REFOJOS; Marina MACHADO CASIMIRO; Carlos José DIAS PEREIRA; Luisa Maria PINHEIRO VALENTE; Daniela OLIVEIRA RESENDE
Original assignee: Ciimar Centro Interdisciplinar De Investigacao Marinha E Ambiental; Instituto Politecnico De Coimbra; Universidade Catolica Portuguesa - Ucp; Universidade do Porto
Current assignee: Ciimar Centro Interdisciplinar De Investigacao Marinha E Ambiental; Instituto Politecnico De Coimbra; Universidade Catolica Portuguesa - Ucp; Universidade do Porto
Priority date: 2020-09-17
Filing date: 2021-09-17
Publication date: 2023-07-26
Also published as: WO2022058962A1

Abstract

The present disclosure relates to method to obtain a new hydrolysate, a new hydrolysate, and uses thereof. Namely an extract from blood hydrolysate (BH), this new extract may be used as a food supplement in aquafeeds to promote fish robustness and control bacterial infections. The extract or composition of the present disclosure may be used in medicine or veterinary, namely in in the prevention or treatment of Tenacibaculum maritimum infections.

Description

D E S C R I P T I O N

N EW HYDROLYSATES TO CONTROL I NFECTIONS, M ETHODS AND

USES THEREOF

TECHNICAL FIELD

[0001] The present disclosure relates to method to obtain a new hydrolysate, a new hydrolysate, preferably blood hydrolysate (BH), that can be used as a food supplement in aquafeeds to promote fish robustness and control bacterial infections, in particular Tenacibaculum maritimum infections.

BACKGROUND

[0002] Bacterial outbreaks, in particular Tenacibaculum maritimum infections, often occur in marine aquaculture farms, with a very high impact on fish health status, resulting in high mortality rates in aquaculture production. Until now, T. maritimum outbreaks have usually been treated by antibiotics, but antibiotic use may lead to the development of resistant bacteria.

[0003] Bioactive peptides are small amino acid chains with interesting properties (beyond their nutritional value), including antioxidant, mineral-binding, immunomodulatory or antimicrobial activities.

[0004] In an embodiment, the present disclosure corresponds to a new and effective way to address fish bacterial infections and represents a good strategy to reduce fish susceptibility to pathogens. Additionally, it promotes fish production in marine fish farms, while also increasing the economic value of animal blood and minimizing waste, contributing to a circular economy approach.

[0005] These facts are disclosed in order to illustrate the technical problem addressed by the present disclosure. GENERAL DESCRIPTION

[0006] The present disclosure relates to method to obtain a new hydrolysate, a new hydrolysate, and uses thereof. Namely an extract from blood hydrolysate (BH), this new extract may be used as a food supplement in aquafeeds to promote fish robustness and control bacterial infections. The extract or composition of the present disclosure may be used in medicine or veterinary, namely in in the prevention or treatment of Tenacibaculum maritimum infections.

[0007] An aspect of the present invention relates to a method to obtain an extract from slaughter industry by-product, the method comprising: obtaining a liquid waste of slaughter industry by-product wherein the main element of the liquid waste is blood; heating the liquid waste in water at a temperature and time suitable to cook the mixture; hydrolysing the cooked liquid waste with Cynara cardunculus L., preferably with 4 (wt.%) of Cynara cardunculus L., at 50 - 60 °C for 4h and pH 5.2; subjecting the mixture of the previous step to a plurality of filtrations, optionally at least a nanofiltration; collecting the retentate or optionally the filtrate; freeze-drying the retentate to obtain the extract in a solid form.

[0008] In an embodiment, the hydrolysing step is performed with Cynara cardunculus L. Preferably the amount of Cynara cardunculus L ranges 2-6 (wt.%). More preferably, the hydrolysing step is performed with 4 (wt.%) of Cynara cardunculus L., preferably at 50-60 °C for 4h and pH 5.2

[0009] In an embodiment, the slaughter industry by-product is blood, preferably swine blood.

[0010] In an embodiment, the filtering step is performed by microfiltration, nanofiltration, or combinations thereof. In a further embodiment, the nanofiltration uses a cut-off membrane threshold of 3 kDa, 120 g mol ^-1, or combinations thereof.

[0011] In an embodiment, the method described in the present disclosure further comprises subjecting a retentate fraction to a reverse osmosis process.

[0012] The present disclosure also relates to an extract obtainable by the method described in the present document, the extract comprising: 20 to 30 wt.% of total proteins; 0.10 to 0.20 wt.% of total fat; and 65 to 75 wt.% of humidity. [0013] In an embodiment, the total microbial count of the extract is less than 7 x 10⁷ CFU mL ¹.

[0014] In another embodiment, the total protein content of the extract ranges from 65 to 90 wt.%. In another embodiment, the protein size ranges from 1200 Da to 28852 Da, preferably 1200 to 14000 Da.

[0015] The present disclosure also relates to an extract as described in previous embodiments for use in medicine or veterinary. Surprisingly, it was found that the administration of the extracts of the present disclosure prevent or treat adverse infections and significantly decreasing mortality (see fig 6).

[0016] In an embodiment, the extract is for use in the prevention or treatment of infections, preferably in the prevention or treatment of fish bacterial infections. In a further embodiment, the fish bacterial infection is caused by Tenacibaculum maritimum.

[0017] An aspect of the present disclosure relates to a composition comprising the extract as described in any of the previous embodiments, preferably a food composition, more preferably an animal food composition.

[0018] In an embodiment, the animal is a fish, preferably wherein the animal is seabass.

[0019] In an embodiment, the extract amount in the composition ranges for 0.1 - 10 % (wt/ wt); preferably 0.5 - 5 % (wt/ wt); more preferably 1- 4 % (wt/ wt); even more preferably 2- 3 % (wt/ wt).

[0020] In an embodiment, the extract/composition of the present disclosure is administrated in a dairy dosage, preferably at least twice a day; more preferably three times a day.

[0021] In an embodiment, the extract/composition of the present disclosure is administrated in a dairy dosage at least for 30 days, preferably for at least 60 days, preferably for at least 70 days.

[0022] In an embodiment, the composition may further comprises a suitable food ingredient, flavours, stabilizers, additives, or combinations thereof. [0023] In an embodiment, the extract/composition of the present disclosure is an extrudable composition.

[0024] In an embodiment, the composition is an oral composition.

[0025] The present disclosure also relates to a food supplemental or nutraceutical composition comprising the composition/extract described in the present disclosure.

[0026] Surprisingly, animals fed with the extract/composition of the present disclosure showed a significantly higher resistance to bacterial infections, in particular infections caused by Tenacibaculum maritimum. As a result, the mortality rate of the animals, in particular fish and more in particular seabass, fed with the extract described in the present disclosure was lower than the animals fed with the commercially available food compositions.

BRIEF DESCRIPTION OF THE DRAWINGS

[0027] The following figures provide preferred embodiments for illustrating the disclosure and should not be seen as limiting the scope of invention.

[0028] Figure 1: A - Schematic representation of the experimental procedure used to obtain the blood extracts: microfiltration retentate (MFR), nanofiltration retentate (NFR), and nanofiltration filtrate (NFF) fractions. B - Photographic representation of the obtained blood extracts, wherein the colour of MFR extract is brown, NFR extract is orange, NFF extract is yellow.

[0029] Figure 2: Total protein content (wt.%) of MFR, NFR and NFF extracts.

[0030] Figure 3: Peptide profile of MFR, NFR and NFF extracts determined by fast protein liquid chromatography (FPLC).

[0031] Figure 4: 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) and oxygen-radical absorbance-capacity assay (ORAC) antioxidant capacity of MFR, NFR and NFF extracts.

[0032] Figure 5: Feed conversion ratio (FCR) and specific growth rate (SGR) of fish fed the experimental diets. [0033] Figure 6: Mortality of fish fed the experimental diets after exposure to T. maritimum infection.

DETAILED DESCRI PTION

[0034] The present disclosure relates to a method to obtain an extract from slaughter industry by-product. The present document also discloses the extract obtainable by the described method and the use of said extract in fish food preparations and/or for the prevention of infections.

[0035] In an embodiment, swine blood was cooked and enzymatically hydrolysed with Cynara cardunculus L.; preferably with 4 (wt.%) of Cynara cardunculus L., during 4 h at 55 °C and pH 5.2. 200 kg of cooked swine blood were processed with 300 kg of water. After hydrolysis, the resultant product was filtered with a sieve (0.5 mm) and the solid (blood cells) and liquid fractions (350 L) were separated.

[0036] In an embodiment, the resultant liquid fraction was separated by microfiltration (MF) with a spiral-wound MF module (0.6 pm; 5.5 m²), resulting in 70 L (with 15.52% of total solids) of MF retentate (MFR) and 280 L of MF filtrate (MFF). The MFR was submitted to reverse osmosis and freeze-dried. The MFF was subsequently submitted to a nanofiltration (NF), recurring to a cut-off membrane threshold of 3 kDa. From the two resultant fractions, the nanofiltration retentate (NFR; 15 L) was submitted to reverse osmosis and freeze-dried. The other fraction, NF filtrate (265 L), was resubmitted to a sequential step of NF with a cut-off membrane threshold of 120 g mol ^-1. The retentate of this stage, NF filtrate (NFF; 50 L with 0.78% of total solids) was also submitted to reverse osmosis and freeze-dried. Three different extracts were obtained - MFR, NFR and NFF. Figure 1-A schematizes this experimental procedure, and Figure 1-B illustrates the different extracts obtained, wherein the colour of MFR extract is brown, NFR extract is orange, NFF extract is yellow.

[0037] In an embodiment, swine blood was obtained as a by-product of meet industries. Pig's bleeding was carried out using a bleeding pig trocart. The blood was collected in a special canister, covered with a plastic bag. The swine blood by-product was followed and traceable for the sample to ensure that only blood from animals considered fit for human consumption was collected. The raw material (swine blood) was analyzed in three different batches and a physical-chemical and microbiological characterization was carried out.

[0038] In an embodiment, the total fat and protein contents as well as humidity and ash were analyzed for three different batches and are present in Table 1. The total fat content was determined by Soxhlet method, after hydrolysis with 4N hydrochloric acid and extraction with petroleum ether. Total protein content was determined by the Kjelda h I method, using a Kjeltec system 1002 distilling unit. The conversion factor used was 6.25. Dry matter and ashes were determined according to NP1614-l:2009 and NP1615:2002 Portuguese standards respectively.

Table 1. Physical-chemical characterization of raw material (swine blood).

Swine blood

Batch Humidity (wt.%) Total protein (wt.%) Total fat (wt.%) Ash (wt.%)

1 70.05 ± 0.07 29.85 ± 0.21 0.20 ± 0.00 0.87 ± 0.01

II 69.55 ± 0.21 29.10 ± 0.14 0.10 ± 0.00 0.86 ± 0.02

III 66.85 ± 0.07 29.65 ± 0.21 0.10 ± 0.00 0.88 ± 0.01

[0039] In another embodiment, microbiological characterization was performed considering total microbial counts, Enterobacteriaceae, and molds and yeasts, being the results present in Table 2.

Table 2. Microbiological characterization (colony-forming unit per mL; CFU mL ^-1) of raw material (swine blood).

Microorganisms Batch 1 Batch II Batch III

Total microbial counts 9.00 x 10⁵ 3.43 x 10⁶ 6.60 x 10⁷

Enterobacteriaceae 1.91 x 10³ 1.66 x 10⁴ 6.38 x 10⁴

Molds and yeasts 9.20 x 10³ - 3.10 x l0³ [0040] In an embodiment, the total protein of the three different extracts was determined by the Kjeldahl method. MFR and NFR extracts had a high total protein content, approximately 90 wt.%, while the NFF showed a value of about 65% (Figure 2).

[0041] In a further embodiment, the protein and peptide profiles were determined by Fast protein liquid Chromatography (FPLC). The protein and peptide profiles of MFR, NFR and NFF were similar, as is present in Figure 3. However, the concentrations of each fraction were very different. All extracts had the highest peaks in the region between 14000 and 1200 Da but the NFF fraction had high content in peptides smaller than 1200 Da (Figure 3), i.e. small peptides.

[0042] In another embodiment, the antioxidant capacity of the obtained extracts was evaluated by 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) and oxygenradical absorbance-capacity assay (ORAC). The NFF fraction showed the greatest antioxidant activity in both methods (Figure 4). The results were in line with the peptide profile, since there was a greater number of peptides with lower molecular weight. NFR fraction had also an interesting antioxidant potential, although lower than that of NFF. The MFR fraction had the lowest antioxidant capacity of all extracts tested.

[0043] In an embodiment, the NFR hydrolysate was included in diets for European seabass (Dicentrarchus labrax). Three isolipidic (16%, as dry matter basis), isoproteic (54%) and isoenergetic (22 kJ/g) diets were studied: a fishmeal (FM) based diet (positive control, PC), a commercial-based diet where 50% of FM was replaced by vegetable proteins (negative control, NC) and a diet where 3% of the NFR was added to the NC, at the expense of plant protein sources (table 3). PC was selected from a premium diet in aquaculture, known to result in high growth. However, this dietary formulation is completely unsustainable due to the high level of marine protein sources (fishmeal). Thus, a more practical diet, mainly relying on plant protein sources, is nowadays used by the seabass farming and was tested as NC and compared with the NFR diet. All diets were extruded by SPAROS Lda. (Olhao, Portugal) under standard conditions. Table 3 - Ingredients of the diets used in this experiment.

Ingredients PC NC NFR wt.%

Fishmeal 25.000 12.500 12.500

Soy protein concentrate 25.000 25.000 25.000

Wheat gluten 10.700 13.500 10.100

Corn gluten 7?500 15.000 15?000

Soybean meal 5.000 10.000 10.000

Wheat meal 12.100 7.240 7.340

Fish oil 13.000 13.400 13.700

Vitamins & Mineral Premix 0.500 0.500 0.500

Dicalcium Phosphate 1.200 2.800 2.800

L-Tryptophan 0 0.060 0.060

NFR 0 0 3.000

[0044] In an embodiment, juvenile European seabass, reared in a commercial fish farm (Acuinuga, S.L., Spain), were transported to the experimental facility. After 15 days of acclimation, fish were fasted for 24h, then individually weighed and measured and finally distributed into a recirculating saltwater system (RAS) composed of 15 fiberglass tanks of 250 L each, at a density of 3.5 kg rrr³. Diets were randomly allocated to triplicate tanks in order to have independent replicates. Each tank was provided with filtered, heated (20 ± 1 °C) and saltwater (36 ± l%o) at a flow rate of 16 L min^-1, under an artificial photoperiod of 12-h light. Physical and chemical water parameters (temperature, salinity, redox potential, pH and concentration of nitrogenous compounds) were monitored during the trial and maintained at optimal levels for this species. Fish were fed 3 times daily until visual satiety, for a period of 74 days. At the end of the growth trial, fish were fasted for 24 h before sampling. All fish were individually weighed and measured for evaluation of growth performance, and feed consumption was registered. For determination of chemical composition, 15 fish from the initial fish stock and 6 fish per tank (at the end of the trial) were collected, sacrificed with anaesthetic overdose (60 pL L’¹ of 2-Phenoxyethanol; Sigma-Aldrich^ MO, USA) and stored at -20 °C until analyses.

[0045] In an embodiment, diets were ground prior to analysis. Dry matter (DM) was evaluated after 24 h at 105 ± 1 °C; ash was determined by combustion in a muffle furnace (Nabertherm L9/11/B170; Germany; 550 °C for 6 h); crude protein (CP) was measured using a Leco nitrogen analyser (Model FP-528, Leco Corporation, St. Joseph, USA); crude fat (CF) by petroleum ether (40-60 °C) extraction (CF, SoxtecTM 2055, Foss, Hbganas, Sweden) and gross energy in an adiabatic bomb calorimeter (Werke C2000, IKA, Staufen, Germany) calibrated with benzoic acid.

[0046] In another embodiment, for the determination of whole-body composition, fish from each tank were ground, pooled and moisture was calculated (105 °C for 24 h). Afterwards, fish were freeze-dried before further analysis of ash, protein and fat content, following the same methods performed for the diets.

[0047] In an embodiment, high mortality rates often occur in marine aquaculture farms due to bacterial infections. Among them, Tenacibaculum maritimum outbreaks are particularly important causing significant loses to marine fish farmers. Until now, T. maritimum outbreaks have usually been treated by vaccination and antibiotics, but current commercial vaccines are not efficient and antibiotic use may lead to the development of resistant bacteria. So, after the growth trial, the resistance of seabass, previously fed the experimental diets for 74 days, towards this pathogen was evaluated. A T. maritimum strain (ACC13.1; serotype 03) isolated from Senegalese sole in a local fish farm (Portugal) was used. Bacteria were cultured at 22 °C in marine agar for 48 h and then inoculated in marine broth for 18 hours. To prepare the inoculum, exponentially growing bacteria were collected and adjusted to a final concentration of 3.5 x 10⁵ colony forming units (cfu) L ¹. By the end of the growth trial, 10 fish from each tank were transferred to new rearing 130 L tanks with strong aeration for inoculation with the bacteria during 2 hours. Subsequently, the water of each tank was changed three times and the recirculation was re-established. Fish were daily supervised, examined for the detection of T. maritimum, and the cumulative mortality of each group was assessed until the 8^th day after infection considering the formula: [(number of dead animals/ number of total animals)*100]. Any survivors at the end of this challenge trial were euthanized by an aesthetic overdose (1 mL L ¹; 2-phenoxyethanol, Sigma).

[0048] In an embodiment, after the 74 days growth trial, the specific growth rate (SGR) and feed conversion ratio (FCR) of fish fed NFR did not differ significantly from those either fed the PC and NC diets (Figure 5). Final body composition and nutrient and energy retention were also similar among diets (Table 4). Regarding the infectious challenge, fish previously fed with the NFR had the lowest mortality rate (<5%) after infection with T. maritimum that differed significantly from the NC group (27%) (Figure 6). NC represents a standard commercial diet used in European seabass farming. Fish fed plant-protein rich diets (like the presently used NC) have often been associated with increased stress and higher susceptibility to diseases compared to fishmeal-based diets (like the PC used here). In fact, the present results clearly showed that fish fed the PC were less susceptible to T. maritimum infection than those previously fed the NC, resulting in a lower mortality. However, the supplementation of the NC diets with NFR hydrolysates resulted in a significant reduction of mortality rate after infection, that was even much lower than that observed in fish fed the PC. The dietary inclusion of NFR hydrolysates is a very promising achievement in the development of fortified diets for European seabass, by increasing fish resistance and survival rate after T. maritimum infection. Overall, results indicate that dietary supplementation with NFR clearly improves the resistance of European seabass to T. maritimum without affecting growth rate, and can hence become a valuable feed additive for this species, contributing to a reduction of the use of antibiotics.

Table 4 - Whole body composition and nutrient retention of European seabass fed the experimental diets.

Dietary treatments

PC NC MFR NFR AH

Final body composition (% wet weight)

DM 36.6 ± 1.5 36.8 ± 1.1 33.6 ± 3.9 35.9 ± 1.1 36.5 ± 0.6

Ash 3.7 ± 0.5 3.7 ± 0.2 4.6 ± 0.4 3.6 ± 0.5 3.7 ± 0.5

Protein 17.2 ± 0.3 17.6 ± 0.5 18.39 ± 1.5 17.2 ± 0.4 17.6 ± 0.4

Lipid 16.0 ± 0.9 15.7 ± 0.9 10.8 ± 2.4 15.2± 0.8 15.2 ± 0.7

Energy 9.6 ± 0.2 9.8 ± 0.3 8.3 ± 1.1 9.4 ± 0.4 9.6 ± 0.2

Retention (%)

DM 33.0 ± 2.2^a 33.4 ± 0.9^a 23.6 ± 4.1^b 31.2 ± 1.6^a 31.0 ± 1.0^a

Protein 27.2 ± 1.3 28.1 ± 0.8 23.1 ± 2.8 26.1 ± 0.7 26.1 ± 0.6 Lipid 100.2 ± 7.7^a 98.9 ± 5.6^a 53.1 ± 16.0^b 90.5 ± 6.4^a 89.4 ± 5.3^a Energy (kJ/g) 40.3 ± 1.4^a 41.0 ± 1.0^a 26.8 ± 5.3^b 37.5 ± 2.3^a 37.3 ± l.l^a

[0049] The term "comprising" whenever used in this document is intended to indicate the presence of stated features, integers, steps, components, but not to preclude the presence or addition of one or more other features, integers, steps, components or groups thereof.

[0050] The disclosure should not be seen in any way restricted to the embodiments described and a person with ordinary skill in the art will foresee many possibilities to modifications thereof. The above-described embodiments are combinable.

[0051] The following claims further set out particular embodiments of the disclosure.

Claims

C L A I M S Method to obtain a protein extract from slaughter industry by-product, comprising the following steps: obtaining a liquid waste of slaughter industry by-product wherein the main element of the liquid waste is blood; heating the liquid waste in water at a temperature and time suitable to cook the mixture; hydrolysing the cooked liquid waste; subjecting the mixture of the previous step to a plurality of filtrations, optionally at least a nanofiltration; collecting the retentate or optionally the filtrate; freeze-drying the retentate to obtain the extract in a solid form. Method according to the previous claim wherein hydrolysing step is performed with Cynara cardunculus L. Method according to the previous claim wherein the amount of Cynara cardunculus L ranges 2-6 (wt.%). Method according to any of the previous claims wherein the hydrolysing step is performed with 4 (wt.%) of Cynara cardunculus L., preferably at 50-60 °C for 4h and pH 5.2 Method according to any of the previous claims wherein the slaughter industry byproduct is blood, preferably swine blood. Method according to any of the previous claims wherein the filtering step is performed by microfiltration, nanofiltration, or combinations thereof. Method according to any of the previous claims further comprising subjecting a retentate fraction to a reverse osmose. Method according to the previous claim wherein the nanofiltration uses a cut-off membrane threshold of 3 kDa, 120 g mol ^-1, or combinations thereof. Extract obtainable by the method described in any of the previous claims comprising:

20 to 30 wt.% of total proteins;

0.10 to 0.20 wt.% of total fat; and

65 to 75 wt.% of humidity. Extract according to the previous claim wherein the total microbial count is less than 7 x lO⁷CFU mL ¹. Extract according to any of the previous claims 9-10 wherein the total protein content ranges from 65 to 90 wt.%. Extract according to any of the previous claims 9-11 wherein the protein size ranges from 1200 Da to 28852 Da. Extract according to any of the previous claims 9-12 wherein the protein size ranges froml200 to 14000 Da. Extract as described in any of the previous claims 9-13 for use in medicine or veterinary. Extract according to previous claim for use in the prevention or treatment of infections. Extract according to previous claim for use in the prevention or treatment of fish bacterial infections. Extract according to previous claim wherein the fish bacterial infection is caused by Tenacibaculum maritimum. Composition comprising the extract as described in any of the claims 9-17 or the extract obtained by the method described in any of the claims 1-8, preferably a food composition, more preferably an animal food composition. Composition according to the previous claim wherein the animal is a fish. Composition according to the previous claim wherein the fish is seabass. Composition according to the previous claims 18-20 wherein the extract amount ranges from 0.1 - 10 % (wt/ wt). Composition according to the previous claims 18-21 wherein the extract amount ranges from 0.5 - 5 % (wt/ wt); more preferably 1- 4 % (wt/ wt); even more preferably 2- 3 % (wt/ wt). Composition according to the previous claims 18-22 wherein the composition is administrated in a dairy dosage, preferably the composition is administrated at least twice a day dose; more preferably the composition is administrated three times a day dose. Composition according to the previous claims 18-23 wherein the composition is administrated in a dairy dosage at least for 30 days, preferably for at least 60 days, preferably for at least 70 days. Composition according to the previous claims 18-24 wherein the composition is an oral composition. Composition according to the previous claims wherein the composition further comprises a suitable food ingredient, flavours, stabilizers, additives, or combinations thereof. Composition according to the previous claims 18-26 wherein the composition is an extrudable composition.

14 Food supplemental or nutraceutical composition comprising the composition described in claims 18-27 or the extract as described in any of the claims 9-17 or the extract obtained by the method described in any of the claims 1-8.

15