Sand worm lyophilisate and uses thereof

Abstract

The invention relates to a lyophilisate or to a powder including i) at least one globin, a globin protomer or an extracellular hemoglobin from annelids, and ii) at least one biological material from annelids that is different from said globin, from said protomer and from said hemoglobin.

Claims

1. A method for improving the yield of fermentation carried out by microorganisms, comprising providing to the microorganisms in a culture medium, a pathogen-free lyophilisate or a powder obtained from whole annelids which have been milled beforehand, wherein the pathogen-free lyophilisate or powder comprises i) at least one extracellular hemoglobin, globin or globin protomer from annelids, wherein said hemoglobin, globin or globin protomer comprises iron, and ii) at least one biological material from annelids that is different from said globin, from said protomer and from said hemoglobin, wherein the annelid is chosen from the Arenicolidae family or the Nereididae family and wherein the pathogen-free lyophilisate or powder is provided in an amount sufficient to improve the yield of fermentation carried out by the microorganisms, and wherein the microorganisms are lactic acid bacteria and the fermentation is lactic acid fermentation.

2. The method of claim 1, wherein the at least one biological material from annelids that is different from said globin, from said protomer and from said hemoglobin is an annelid coproduct.

3. The method of claim 2, wherein the annelid coproduct is a worm fraction remaining after extraction of hemoglobin from the annelid.

4. The method of claim 1, wherein the culture medium comprises at least sugar, yeast extract and minerals.

5. The method of claim 1, wherein the microorganisms are in a bioreactor.

6. A method for improving the yield of fermentation carried out by microorganisms, consisting essentially of providing to the microorganisms, in a culture medium, a pathogen-free lyophilisate or a powder obtained from whole annelids which have been milled beforehand, wherein the pathogen-free lyophilisate or powder comprises i) at least one extracellular hemoglobin, globin or globin protomer from annelids, wherein said hemoglobin, globin or globin protomer comprises iron, and ii) at least one biological material from annelids that is different from said globin, from said protomer and from said hemoglobin, wherein the annelid is chosen from the Arenicolidae family or the Nereididae family and wherein the pathogen-free lyophilisate or powder is provided in an amount sufficient to improve the yield of fermentation carried out by the microorganisms, and wherein the microorganisms are lactic acid bacteria and the fermentation is lactic acid fermentation.

Description

EXAMPLE 1: PREPARATION AND ASSAYING OF LYOPHILISATES ACCORDING TO THE INVENTION

Materials & Methods

(1) Worm Powders

(2) The products tested are powders of Arenicola marina and of Nereis virens.

(3) These powders were obtained by lyophilization and milling.

(4) The following media were tested: MEDIUM 1: milliQ H.sub.2O MEDIUM 2: Extraction buffer (400 mM NaCl, 2.95 mM KCl, 32 mM MgSO.sub.4. 7H.sub.2O, 11 mM CaCl.sub.2, 50 mM Tris base, 10 mM ascorbic acid) MEDIUM 3: 1N NaOH (used to redisperse the Hemin, hemoglobin of bovine origin).

Method

(5) 1 g of powder was weighed into a 15 ml centrifuge tube. 3 ml of solution were added and then homogenized, firstly manually and then with a vortex. Photographs were taken of this mixture. This mixture was then centrifuged at 5000 g for 10 minutes. The pellet and the supernatant were then photographed. The supernatant was recovered and frozen at −80° C. Given the small volumes recovered, the experiment was repeated, but on 3 g of powder and 9 ml of solution in 50 ml centrifuge tubes. Aliquots were frozen at −80° C.

(6) For the analytical tests, fresh material was generated in greater amount 3 g of worm powder were weighed out and then resuspended in 15 ml of the redispersion solution. The material was then centrifuged and the supernatant was recovered and then filtered through 0.8 μm and 0.22 μm filters. The functionality and the purity were determined and an assay was carried out on the samples.

Results

(7) 1. Lyophilization

(8) For 5 kg of Arenicola, 670 g of powder were generated, i.e. a factor of 7.5.

(9) For 6 kg of Nereis, 921 g of powder were generated, i.e. a factor of 6.5.

(10) 2. Analytical Results

(11) a) Assay

(12) The hemoglobin concentrations are between 3 and 8 g/kg of fresh worms.

(13) TABLE-US-00001 Arenicola Nereis Water Tris buffer Extraction buffer Water Tris buffer Extraction buffer [M101] in g/l with a 8 8 11 4 7 9 ⅕ dilution [M101] in g/kg of worm 38 40 56 22 36 47 powder [M101] in g/kg of fresh 5 5 8 3 6 7 worms

(14) b) Conductivity

(15) The conductivity of the solids is high (˜130 mS/cm).

EXAMPLE 2: TESTS WITH THE LYOPHILISATES ACCORDING TO THE INVENTION

Materials & Methods

(16) Worm Powders

(17) HEMARINA A: from Arenicola marina

(18) HEMARINA B: from Nereis virens

(19) The powders were obtained as in example 1.

(20) Sample Preparation i) Flask culture: dissolution of the powders at 200 g/l then filtration of the supernatant and addition to the flasks. ii) Bioreactor culture: direct addition to the reactors in a proportion of 1 g/l of powder before autoclaving.

(21) Culture Conditions i) Flask (100 ml, 0.5 and 11) culture:

(22) The strain used is a strain of Lactococcus lactis SB50.

(23) The tests were carried out overnight (16-18 h) at 30° C. with shaking at 180 rpm.

(24) The composition of the culture medium for the strain is the following: glucose (1%), maltose (0.1%), yeast extract (2%) and minerals.

(25) The culture conditions are the following: filling to 10% useful volume; a positive control with addition of hemin (hemoglobin of bovine origin) at 10 ppm final concentration is used.

(26) The HEMARINA samples (powders in solution) were added post-sterilization after centrifugation. ii) Bioreactor (11 useful volume) culture:

(27) The tests were carried out overnight (15-16 h) at 30° C.

(28) The composition of the culture medium for the strain is identical to that of the flasks, with an addition of 0.01% antifoam.

(29) The culture conditions are the following: filling to 60% useful volume, pO.sub.2 regulated at 20% or 80% and pH regulated at 6.

(30) 1 g/l of powder are added directly to the culture medium before autoclaving.

(31) The optical density (OD) at 600 nm is measured for each culture.

Results

(32) i) Flask Culture:

(33) The ODs are the following:

(34) TABLE-US-00002 Reference OD at 600 nm Control (Lc lactis alone) 1.52 Positive control (Lc lactis + hemin 4.5 10 ppm)

(35) The active concentration of each product (HEMARINA A and HEMARINA B) is equivalent to that of hemin, i.e. is approximately 0.25 ppm.

(36) ii) Bioreactor Culture:

(37) The results are the following:

(38) TABLE-US-00003 Control Positive control HEMARINA A OD at 600 nm 10.63 10.9 12.07 Dry weight (g/l) 2.8 3.4 4.1 Volume NH.sub.4OH 18.2 12.3 15.8 (ml) Viability (CFU/ml) 1.75E+09 5.03E+09 6.42E+09

(39) The viability of the strain is improved three- to four-fold in the presence of the HEMARINA A or HEMARINA B products.

(40) The production of lactic acid (corresponding to the volume of NH.sub.4OH produced) is less than the control but more than the positive control (hemin).

(41) The acidifying capacity of the HEMARINA A and HEMARINA B products is equivalent to that of the positive control.

(42) In conclusion, it appears that the HEMARINA A and HEMARINA B products make it possible to improve the viability of lactic acid strains, and have a very good acidifying capacity.

(43) These products could replace bovine and porcine hemins, which are widely used in the food-processing fields, but which have potential risks of viral or prion contamination in humans, and which are not suitable for the production of kosher and/or halal products.