Biomass of the microalgae Schizochytrium mangrovei and method for preparing same

Abstract

The invention concerns a strain of Schizochytrium mangrovei, filed on 22 Nov. 2012 with the CNCM as number I-4702, having the ability to produce a high quantity of docosahexaenoic acid (or DHA) and palmitic acid, the methods for producing the corresponding biomass containing said lipid compounds of interest, and the biomass containing the products and compositions prepared from this strain.

Claims

1. A method for producing a biomass of microalga containing lipid compounds of interest, the method comprising the steps of culturing a strain of Schizochytrium mangrovei deposited on Nov. 22, 2012 at the CNCM under number I-4702 and recovering the biomass rich in lipid compounds of interest, wherein the recovered biomass has between 35 and 40 wt % of DHA, expressed by weight of total fatty acids and between 40 and 50 wt % of palmitic acid, expressed by weight of total fatty acids.

2. The method as claimed in claim 1, characterized in that the biomass is prepared by the sequence of the following steps: culturing the strain in heterotrophic conditions so as to produce a biomass having between 35 and 40 wt % of DHA, expressed by weight of total fatty acids and between 40 and 50 wt % of palmitic acid, expressed by weight of total fatty acids, collecting the biomass thus prepared, and drying said biomass.

3. A method for preparing compositions intended for the food sector comprising adding a biomass produced by the method of claim 1 to the compositions.

4. The method as claimed in claim 3, wherein said biomass comprises: between 35 and 40 wt % of DHA, expressed by weight of total fatty acids; between 40 and 50 wt % of palmitic acid, expressed by weight of total fatty acids; and between 1.5 and 2 wt % of phospholipids, expressed by weight of biomass at 99% of dry matter.

Description

EXAMPLE 1

Production of a Biomass Rich in DHA and Palmitic Acid by the Schizochytrium mangrovei Strain CNCM I-4702

(1) The compositions of the culture media and the fermentation conditions are given in the following tables.

(2) TABLE-US-00002 TABLE 1 Composition of the culture media Successive fermentors of: 100 liters 1 m.sup.3 10 m.sup.3 Effective volume of the fermentor 70 liters 700 liters 7000 liters Glucose (kg) 6 41.3 1120 Monosodium gluconate (kg) 4.494 26.67 / Liquid corn steep (kg) / / 119 Yeast extracts (kg) 0.448 2.03 42 NaCl (kg) 1.4 2.66 16.8 KH.sub.2PO.sub.4 (kg) 0.448 2.80 33.6 MgSO.sub.4 (kg) 1.6 7.35 42 CaCl.sub.2 (kg) 0.02 0.14 4.2 NaHCO.sub.3 (kg) 0.2 0.07 4.2 Antifoam (kg) 0.112 1.12 11.2 (DOW FAX DF 104) Na.sub.2SO.sub.4 (kg) 0.02 0.07 42 Urea (kg) / / 18.9 KCl (kg) / / 2.8 Aqueous ammonia / / 33.6 (28%, liter) “Red” liquid (liter) 0.098 0.98 14 “Green” liquid (liter) 0.140 1.4 19.60

(3) The initial glucose concentration in the sterilized culture medium of the 10 m.sup.3 fermenter is fixed at 15 to 16 g/I.

(4) TABLE-US-00003 TABLE 2 Parameters for control of the preculture and of the 3 successive fermentations Preculture 100 liters 1 m.sup.3 10 m.sup.3 Load volume 200 ml × 3 70 liters 0.7 m.sup.3 7 m.sup.3 Temperature (° C.) 28° C. 28° C. 28° C.  0-68 h: 28° C. 68-80 h: 26° C. pH No No No regulation Regulation 6.2~6.6 Air flow rate (m.sup.3/h) — 4.5-5.0 41-42  0-24 h: 95-105; 24-60 h: 50-60; 60-80 h: 25-30 Pressure (Mpa) — 0.025-0.03  0.025-0.03  0.03 Shaking (rpm) 180 rpm 120-140 140 70-75 Fermentation time (h) 48 24-48 24-48 ~80   

(5) The principle for feeding with glucose during the fermentation in 10 m.sup.3 is the following: the concentration of the glucose feed solution is 50-60%, and when the residual glucose concentration (RCS) decreases to 1 g/l before 48 h, a solution containing 0.65 g/l of glucose is fed in one step until the glucose is exhausted and the fermentation is complete.

(6) The results of the various fermentations are given in the following tables.

(7) TABLE-US-00004 TABLE 3 First fermentation Time (h) 0 8 16 23 pH 5.6 5.6 7.0 7.6 Residual glucose 5.4 / / 4.7 concentration (g/100 ml) Amino nitrogen 269 / / 123 (mg/100 ml) Phosphorus (ppm) 692 / / 159.1 Dry weight of cells (g/l) ~0 / / 29

(8) TABLE-US-00005 TABLE 4 Second fermentation Time (h) 0 6 h 10 h 14 h 18 h 22 h 24 h pH 5.4 7.1 7.6 8.0  8.1 8.05 7.9 Residual glucose 5.8 / / / / / 2.2 concentration (g/100 ml) Amino nitrogen 166 / / / / / 75 (mg/100 ml) Phosphorus (ppm) 725.8 / / / 141.2 / 86.7 Dry weight of cells 1.6 / / / / / 46.7 (g/l)

(9) TABLE-US-00006 TABLE 5 Third fermentation Time (h) 0 12 24 36 48 60 64 72 81 pH 6.7 6.3 5.8 6 6.2 6.45 6.5 6.5 6.6 Residual 14.1 12.1 9.1 11.2 9.6 5.8 5.8 4.35 1.35 glucose concentration (g/100 ml) Amino 165 84 53 36 35 39 / 37 31 nitrogen (mg/100 ml) Air flow rate 95-100 m.sup.3/h 60 m.sup.3/h 30 m.sup.3/h (m.sup.3/h) Temperature 28 26 (° C.) Phosphorus 724.1 692.4 661.3 544.3 599 659 / 651.2 618 (ppm) Dry weight of 11.8 25.7 37.4 38.5 44.2 69.9 / 75.4 80.5 cells (g/l) DHA content / / / 30.7 27.6 34 / 32.75 31.4 (%)

(10) Almost 70% of the amino nitrogen is consumed during the first 24 h of fermentation; the phosphorus is also consumed during the cell growth step and is no longer consumed subsequently (in connection with the regulation of the aeration flow rate and the fermentation temperature).

(11) The level of lipid accumulation and the level of DHA production gradually increase and reach a maximum at 72 hours.

(12) The recovery of the cells is therefore optimal as soon as this fermentation time is reached.

(13) It is chosen to stop the fermentation at 81 h.

EXAMPLE 2

Recovery and Conditioning of the Biomass of the CNCM I-4702 Strain for Applications in Animal and Human Nutrition

(14) The biomass recovered at the end of the fermentation described in example 1 has the following composition:

(15) TABLE-US-00007 TABLE 6 Volume recovered Dry weight of DHA content (m.sup.3) cells (g/l) (%) 7.5 80.5 31.4

(16) The biomass recovered is centrifuged a first time at 6000 g, and the cells recovered are then diluted in sterile water (1.5/1 ratio) and then centrifuged a second time.

(17) It is then subjected to a heat treatment at 70° C. for 15 minutes.

(18) 2.55 t of wet biomass (16.7% of dry matter) are recovered.

(19) The following are added thereto for the formulations intended for animal nutrition: 2% of maltodextrin with a DE (dextrose equivalent) of 18, 0.5% of monoglycerides and diglycerides, 1% of citric acid, 0.2% of anthracyne 2727 (as antioxidant), and 0.2% of tricalcium phosphate.

(20) For the formulations intended for human nutrition, food-grade antioxidants of tocopherol type or extracts of rosemary are used.

(21) This biomass is spray-dried in a single-stage spray dryer (conventional running known to those skilled in the art) in the conditions given in the following Table 7:

(22) TABLE-US-00008 TABLE 7 Parameters Values Solution temperature  65-70° C. Air input temperature 155-160° C. Air output temperature 75° C.-80° C. Pressure ~7.5 Mpa Wet biomass input (kg) 690.7 Dry matter of the biomass (%) 16.7 Theoretical weight of cells (kg) 115.35 Weight of dry cells obtained (kg) 144.03 Drying yield (%) 125

(23) The composition of the dried biomass is the following (Table 8):

(24) TABLE-US-00009 TABLE 8 Parameters Values DHA content (% 35.2 relative to total fatty acids) Proteins N6.25 16.7 in g/100 g crude Phospholipids 1.6 (%) Residual water 1.2 content (%) Ash (%) 6.4 POV (meq/kg) 0.4 P- Anisidine (%) 23.8

EXAMPLE 3

Comparative Study of the Lipid Profile of a Biomass in Accordance with the Invention Compared with Those that are Commercially Available

(25) The fatty acids were determined by gas chromatography in the form of methyl esters after transesterification with methanolic hydrochloric acid and extraction with chloroform. The results are expressed as % distribution; the analysis is carried out by the internal standardization method.

(26) A chromatograph (Varian 3800) equipped with a split-splitless injector with a tapfocus liner and a flame ionization detector was used.

(27) An internal standard solution containing about precisely 0.5 mg of methyl heptadecanoate per ml of methanol was prepared. The methyl heptadecanoate served as a chromatographic point of reference.

(28) About precisely 30 mg of pre-dried sample were weighed into a 6 ml tube. 1 ml of the internal standard solution and then 2 ml of 3N methanolic hydrochloric acid were added using a pipette with two measurement lines. The tube was then stoppered and placed in a dry bath thermostated at 110° C. for 4 h.

(29) After cooling, about 0.5 ml of water and 0.5 ml of saturated aqueous sodium chloride solution were added, and extraction was carried out with 3 times 1 ml of chloroform. The chloroform phases were recovered in a 6 ml tube with them being dried on a column containing sodium sulfate. They were concentrated under a nitrogen stream to about 1 ml and injected.

(30) The % distribution of each fatty acid (i) was obtained by the ratio of the area of the peak of this fatty acid relative to the sum of the areas of all the peaks pinpointed on the chromatogram, from lauric acid (C12:0) to DHA (C22:6 Δ4c, 7c, 10c, 13c, 16c, 19c) inclusive, with the methyl heptadecanoate peak being excluded.

(31) The phospholipids are analyzed after disruption and cold extraction of the biomass, carried out under the following conditions.

(32) Disruption of the Biomass

(33) Precisely 200 mg of fresh biomass are weighed into a screw-top Pyrex tube. About 1-1.5 cm of glass beads (Retsch, reference 22.222.0003) and 0.1 ml of methanol are added. The tube is hermetically closed and stirred by means of a vortex mixer for at least 5 min.

(34) Cold Extraction

(35) Precisely 2 mg of triphenyl phosphate (purity ≥98%) are weighed into a small aluminum boat using a microgram balance.

(36) The boat is placed in a Pyrex NMR tube 5 mm in diameter along with 0.9 ml of methanol and 2 ml of chloroform. The tube is hermetically closed and stirred by means of a vortex mixer for 1 min.

(37) The tube is placed in the refrigerator. After settling out (minimum of 1 hour), the clear upper phase is carefully recovered and is transferred into a glass jar for evaporation to dryness, at ambient temperature, under a nitrogen stream.

(38) The solid extract is dissolved in 0.5 ml of CDCl.sub.3 and 0.1 ml CD.sub.3OD and transferred into an NMR tuba.

(39) In order to express the phospholipid content on the basis of the phosphorus content obtained by NMR, the phosphorus provided by the four main phospholipids is taken into account and oleic acid is used to calculate the molar mass of each of them.

(40) The phospholipid content is equal to the sum of the amounts of these four phospholipids thus calculated. The biomasses analyzed according to these methods (in the following Tables 9 and 10), in addition to that of the invention, are biomasses sold by Aquafauna Bio-Marine Inc., DSM/Martek and New Horizon.

(41) TABLE-US-00010 TABLE 9 Biomass in OMEGA accordance VIE with ALGAMAC NEW example 2 3050 DHA Gold HORIZON Total fatty acids in g/100 g crude and as % relative to total fatty acids g/100 g % g/100 g % g/100 g % g/100 g % Lauric C12:0 0.1 0.2 0.2 0.3 0.2 0.3 <0.1 0.1 Myristic C14:0 2.9 5.4 5.4 10.1 6.1 10.8 1 2.7 Pentadecylic C15:0 0.3 0.5 0.2 0.4 0.2 0.4 <0.1 0.2 Palmitic C16:0 24.3 44.5 12.0 22.3 12.9 23.1 17.4 47.6  Palmitoleic C16:1 Δ9c 0.1 0.3 0.1 0.2 0.1 0.2 <0.1 0.2 Stearic C18:0 0.8 1.4 0.3 0.5 0.3 0.5 0.6 1.6 Oleic C18:1 Δ9c w9 <0.03 <0.03 <0.03 0.2 0.5 Linoleic (LA) C18:2 Δ9c, 12c w6 <0.1 <0.1 <0.03 <0.03 0.2 0.5 g-linolenic (GLA) C18:3 Δ6c, 9c, 12c w6 <0.1 <0.1 0.1 0.2 0.1 0.2 <0.03 — a-linolenic (ALA) C18:3 Δ9c, 12c, 15c w3 <0.1 0.1 <0.1 <0.1 <0.1 <0.1 0.1 0.3 Arachidic C20:0 <0.1 0.1 <0.1 0.1 <0.1 0.1 <0.1 0.1 Stearidonic (SDA, STD) 0.1 0.2 0.2 0.3 0.2 0.3 <0.1 0.2 C18:4 Δ6c, 9c, 12c, 15c w3 Gondoic C20:1 Δ11c w9 <0.03 <0.03 <0.03 <0.03 — Dihomo-gamma-linolenic acid (DGLA) <0.1 0.1 0.2 0.3 0.2 0.3 <0.1 0.1 C20:3 Δ8c, 11c, 14c w6 Arachidonic (AA) C20:4 Δ5c, 8c, 11 c, 14c w6 <0.1 0.1 0.2 0.4 0.2 0.4 <0.1 0.1 (ETE) C20:3 Δ11c, 14c, 17c w3 <0.03 <0.03 <0.03 <0.03 — Behenic C22:0 <0.1 0.1 0.1 0.2 0.1 0.2 <0.1 0.1 Timnodonic EPA 0.2 0.3 0.6 1.0 0.6 1.0 0.2 0.6 C20:5 Δ5c, 8c, 11c, 14c, 17c w3 Lignoceric C24:0 <0.03 0.1 0.2 0.1 0.2 <0.03 — Osbond acid C22:5 Δ4c, 7c, 10c, 13c, 16c w6 4.3 7.9 8.1 15 8.0 14.3 2.7 7.4 Nervonic C24:1 Δ15c w9 <0.1 <0.1 0.1 0.2 0.1 0.2 <0.03 — Clupanodonic DPA <0.1 0.1 0.2 0.3 0.2 0.3 <0.1 0.1 C22:5 Δ7c, 10c, 13c, 16c, 19c w3 Cervonic DHA 19.2 35.2 23.6 43.9 24.3 43.3 12.8 35   C22:6 Δ4c, 7c, 10c, 13c, 16c, 19c w3 Others <3.4 <4.1 <3.9 2.6 Total fatty acids 53 52 54

(42) TABLE-US-00011 TABLE 10 Biomass in accordance OMEGA with the invention ALGAMAC DHA VIE NEW according to example 1 3050 GOLD HORIZON Phospholipids in g/100 g crude, 1.6 0.8 0.9 0.6 base C18:1 Phosphatidylcholine as %/crude 1.1 0.6 0.7 0.5 Lysophosphatidylcholine as %/crude 0.2 0.1 0.1 0.1 Phosphatidylethanolamine as %/crude 0.3 0.1 0.1 <0.1 Phosphatidylglycerol as %/crude <0.1 <0.1 0 <0.1 Dry matter in g/100 g 99.1 98.0 98.5 97.2 Ash in g/100 g crude 6.4 9.5 10.0 Nitrogen N6.25 in g/100 g crude 16.7 12.4 13.2 3.6

(43) It appears, on reading the results presented here, that relative to the fatty acids profile: the biomass according to the invention has a DHA content slightly lower than that of the commercial DHA-rich biomasses, but a palmitic acid content which is double, which constitutes as it were the fingerprint of the biomass of Schizochytrium mangrovei of the invention; compared with commercial oils that have an equivalent DHA content and palmitic acid content, the biomass according to the invention has double the content of phospholipids (more particularly of phosphatidylcholine); and a much higher amino nitrogen content.