U.S. patent application number 14/910884 was filed with the patent office on 2016-07-07 for method for optimising the production efficiency, organoleptic quality and stability over time of a protein-rich microalgae biomass.
The applicant listed for this patent is ROQUETTE FRERES. Invention is credited to AMANDINE DRUON, SAMUEL PATINIER, BEATRICE TOURSEL.
Application Number | 20160194598 14/910884 |
Document ID | / |
Family ID | 51352716 |
Filed Date | 2016-07-07 |
United States Patent
Application |
20160194598 |
Kind Code |
A1 |
DRUON; AMANDINE ; et
al. |
July 7, 2016 |
METHOD FOR OPTIMISING THE PRODUCTION EFFICIENCY, ORGANOLEPTIC
QUALITY AND STABILITY OVER TIME OF A PROTEIN-RICH MICROALGAE
BIOMASS
Abstract
The present invention relates to a method for optimising the
downstream processing of a protein-rich microalgac biomass of the
Chlorella genus previously prepared by fermentation in
heterotrophic conditions and in the absence of light, comprising:
1) providing biomass comprising more than 50% protein by dry weight
of biomass; next, at low temperature, carrying out the following
steps: 2) harvesting the biomass at the end of fermentation, 3)
washing and concentrating the biomass, 4) optionally, lysing the
biomass, next, without low temperature stress, 5) optionally,
concentrating the biomass slurry, 6) applying heat treatment, 7)
drying the biomass obtained in this way in order to obtain the
product, a step of adjusting the pH to 7 being applied before or
after the heat treatment step 6).
Inventors: |
DRUON; AMANDINE; (LILLE,
FR) ; PATINIER; SAMUEL; (QUESNOY-SUR-DEULE, FR)
; TOURSEL; BEATRICE; (GONNEHEM, FR) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
ROQUETTE FRERES |
Lestrem |
|
FR |
|
|
Family ID: |
51352716 |
Appl. No.: |
14/910884 |
Filed: |
July 24, 2014 |
PCT Filed: |
July 24, 2014 |
PCT NO: |
PCT/FR2014/051918 |
371 Date: |
February 8, 2016 |
Current U.S.
Class: |
435/25 ;
435/257.3; 435/29 |
Current CPC
Class: |
C12N 1/005 20130101;
C12N 1/066 20130101; C12N 1/12 20130101; C12N 1/02 20130101 |
International
Class: |
C12N 1/12 20060101
C12N001/12; C12N 1/06 20060101 C12N001/06 |
Foreign Application Data
Date |
Code |
Application Number |
Jul 25, 2013 |
FR |
13 57326 |
Mar 25, 2014 |
FR |
14 52486 |
Claims
1-10 (canceled).
11. A method for optimizing the downstream processing of a
protein-rich biomass of microalgae of the Chlorella genus which has
been prepared beforehand by fermentation in heterotrophic
conditions and in the absence of light, comprising: 1) providing a
biomass comprising more than 50% proteins by dry weight of biomass;
then, at low temperature: 2) recovering the biomass at the end of
fermentation, 3) washing and concentrating the biomass, 4)
optionally lysing the biomass, then, with no low temperature
constraints: 5) optionally concentrating the biomass suspension, 6)
applying a heat treatment, and 7) drying the resulting biomass to
obtain the product, a step of adjusting the pH to 7 being applied
before or after step 6) of heat treatment.
12. The method as claimed in claim 11, characterized in that the
protein comprises more than 60% by dry weight of the biomass.
13. The method as claimed in claim 11, characterized in that the
heat treatment is a high temperature/short time (HTST) heat
treatment for 30 seconds to 5 minutes at a temperature lower than
100.degree. C.
14. The method as claimed in claim 11, characterized in that the
heat treatment is an ultra-high temperature (UHT) heat treatment at
a temperature of between 100.degree. C. and 150.degree. C. for 5 to
30 seconds.
15. The method as claimed in claim 11, characterized in that the
biomass is washed with at most 6 volumes of water per 1 volume of
biomass.
16. The method as claimed in claim 11, characterized in that the
biomass suspension is neutralized to pH 7 by adding KOH or
NaOH.
17. The method as claimed in claim 11, characterized in that the
cells of the biomass are lysed by milling.
18. The method as claimed in claim 11, characterized in that the
biomass is concentrated by centrifugation or evaporation.
19. The method as claimed in claim 11, characterized in that the
effects of the steps of processing the microalgal biomass on the
quality of the product are also determined by one or more of the
following parameters: measuring the dry cell weight in the biomass;
measuring the sugar content; determining the amount of proteins;
analyzing the volatile organic compounds; measuring enzyme
activities, in particular lipoxygenase activity; measuring the
coloration or the pigment content; measuring the content of metals,
in particular iron, copper or nickel; and/or determining the degree
of oxidation.
20. The method as claimed in claim 11, characterized in that the
microalgae of the Chlorella genus are selected from the group
consisting of Chlorella vulgaris, Chlorella sorokiniana and
Chlorella protothecoides.
21. The method as claimed in claim 20, characterized in that the
microalgae of the Chlorella genus is Chlorella protothecoides.
22. The method as claimed in claim 17, characterized in that the
cells of the biomass are lysed by bead milling.
23. The method as claimed in claim 15, characterized in that the
biomass is washed with at most 3 volumes of water per 1 volume of
biomass.
24. The method as claimed in claim 12, characterized in that the
protein comprises more than 65% by dry weight of the biomass.
25. The method as claimed in claim 12, characterized in that the
protein comprises more than 70% by dry weight of the biomass.
Description
[0001] The present invention relates to a method for optimizing the
production efficiency, the organoleptic quality and the stability
over time of a protein-rich microalgal biomass, said microalgae
being of the Chlorella genus, more particularly Chlorella vulgaris,
Chlorella sorokiniana or Chlorella protothecoides.
PRESENTATION OF THE PRIOR ART
[0002] It is well known to those skilled in the art that chlorellae
are a potential source of food, since they are rich in proteins and
other essential nutrients.
[0003] They contain in particular 45% proteins, 20% fats, 20%
carbohydrates, 5% fibers and 10% minerals and vitamins.
[0004] The use of microalgae (and mainly their proteins) as
foodstuff is being increasingly considered in the search for
alternative sources to meet the increasing global demand for animal
proteins (as reported by the FAO).
[0005] Moreover, the European Union has been suffering from a
structural deficit in plant proteins for years now, which has
amounted in recent years to more than 20 million tons of soy
equivalent, currently imported from South America.
[0006] The mass production of certain protein-rich microalgae is
thus envisioned as a possible way to reduce this "protein
deficit".
[0007] Extensive analyses and nutritional studies have shown that
these algal proteins are equivalent to conventional plant proteins,
or even are of superior quality.
[0008] Nonetheless, due to the high production costs and technical
difficulties in incorporating the material derived from microalgae
into organoleptically acceptable food preparations, the widespread
distribution of microalgal proteins is still in its infancy.
[0009] Microalgal biomasses from various species having a high
percentage of proteins have been reported (see table 1 in Becker,
Biotechnology Advances (2007), 25:207-210).
[0010] Additionally, a certain number of patent applications in the
prior art, such as patent application WO 2010/045368, teach that it
is possible to adjust the culturing conditions so as to further
increase the protein content of the microalgal biomass.
[0011] Preferably, for the microalgae which have this capacity, the
culturing is carried out heterotrophically, in the absence of light
and in the presence of an assimilable source of carbon.
[0012] These routes for heterotrophic growth make it possible both
to mass-produce microalgae and to improve the organoleptic quality
thereof by inhibiting the synthesis by the microalga of
chlorophyll, which is the source of the pronounced green tea flavor
in food preparations containing same.
[0013] To enrich the protein content, the microalga is cultured in
a nitrogen-enriched medium in the presence of an abundant source of
carbon such as glucose. In this case, it is of no concern whether
the nitrogen is provided by organic or inorganic sources.
[0014] The microalgal biomass produced in this way typically
contains at least 40%, or even up to 50-60% proteins by dry cell
weight.
[0015] However, nothing should be taken for granted in the sense
that, as well as the work carried out for the upstream processes
for producing protein-rich biomasses--in particular research into
suitable fermentation conditions--other difficulties arise from the
downstream processing of said biomass to incorporate it into food
preparations of interest.
[0016] Conventionally, the downstream processing comprises several
steps: [0017] collecting the microalgae separated from their growth
medium, [0018] pasteurization and washing, [0019] optionally
breaking the cells open to release the molecules of interest from
them, [0020] drying.
[0021] The first step of collecting the cells is carried out using
one or more steps of solid/liquid separation.
[0022] The biomass is usually collected by sedimentation,
centrifugation or filtration, and sometimes an additional
flocculation step is necessary.
[0023] Following this first step, which may enable the biomass to
be concentrated by 50 to 200 times, the microalgal suspension must
be processed rapidly, otherwise it will rapidly break down.
[0024] A first operation consists in pasteurizing said suspension,
that is to say heating it so as to limit or inhibit the microbial
load (growth of contaminating bacteria) but also so as to
inactivate certain enzymes liable to cause undesirable odors or
flavors ("off flavors").
[0025] This operation is conventionally carried out at high
temperature for a short time (what is referred to as a "high
temperature/short time"--HTST--or ultra-high
temperature--UHT--process).
[0026] A second operation is washing, recommended on intact cells
(with volumes of distilled or deionized water) so as to eliminate
soluble impurities.
[0027] In the event that a step of breaking open or rupturing the
cells is envisioned, several routes are possible: mechanical
(homogenizers, bead milling, ultrasonic milling) or non-mechanical
(alkaline route, cycles of freezing/thawing, organic solvents or
osmotic shock).
[0028] The method is chosen as a function of the nature of the
microalgal cell wall which is to be broken, and the nature of the
product which is to be isolated.
[0029] The last downstream treatment step consists in dehydrating
said suspension (intact or lyzed cells). Several methods have been
employed to dry microalgae of the Chlorella, Scenedesmus and
Spirulina genera. The most conventional are spray drying, drying on
a drying drum, and freeze drying. Spray drying is the method most
often used on an industrial scale.
[0030] However, the sensitivity of certain biomasses to oxidation
makes the addition of antioxidants necessary.
[0031] Despite this diversity in methods and in combinations of
methods suitable for microalgae, there remain six main difficulties
(listed a) to f) below) which have not yet been satisfactorily
resolved by those skilled in the art, in particular: [0032] a) the
loss of protein yield during the downstream processing, [0033] b)
the regrettable loss of protein content after the step of heat
deactivation of the biomass (which may result in up to 25% loss),
[0034] c) the generation of uncontrolled undesirable flavors or
odors (off notes) despite the recommended washing steps, [0035] d)
the as yet inexplicable absence of stability over time of the
batches produced, and of reproducibility of stability, since some
batches are stable while others are not, [0036] e) the risks of
microbial contamination of the final product, and [0037] f) the
drop in overall energy efficiency of the purification process,
based on poor management of the biomass concentration factor.
SUMMARY OF THE INVENTION
[0038] To overcome these drawbacks, the applicant company has
chosen to undertake work to implement suitable downstream
processing steps, the efficiency of which can be measured: [0039]
a) by calculating the protein content and yield of the biomass
produced and/or the content of dry biomass, but also [0040] b) by
methods of sensory analysis, and/or [0041] c) of measuring the
stability over time of the batches produced, by virtue of a quite
unique accelerated aging method developed by the applicant
company.
[0042] The present invention relates to a method for optimizing the
downstream processing of a protein-rich biomass of microalgae the
Chlorella genus which has been prepared beforehand by fermentation
in heterotrophic conditions and in the absence of light,
comprising: [0043] 1) providing a biomass comprising more than 50%
proteins by dry weight of biomass; [0044] then, at low temperature:
[0045] 2) recovering the biomass at the end of fermentation, [0046]
3) washing and concentrating the biomass, [0047] 4) optionally
lyzing the biomass, [0048] then, with no low temperature
constraints: [0049] 5) optionally concentrating the biomass
suspension, [0050] 6) applying a heat treatment, [0051] 7) drying
the resulting biomass, to obtain the product, [0052] a step of
adjusting the pH to 7 being applied before or after step 6) of heat
treatment.
[0053] Preferably, the biomass comprises more than 50% by dry
weight of proteins, preferably more than 55%, more preferably still
more than 60, 65 or 70%.
[0054] When the steps are carried out at low temperature, the
temperature is kept lower than 8.degree. C., preferably lower than
4.degree. C. Preferably, this low temperature is applied throughout
steps 2) to 4) of the method in accordance with the invention.
[0055] Preferably, the heat treatment is a high temperature/short
time (HTST) heat treatment for 30 seconds to 5 minutes at a
temperature lower than 100.degree. C.
[0056] Alternatively, the heat treatment is an ultra-high
temperature (UHT) heat treatment at a temperature of between
100.degree. C. and 150.degree. C. for 5 to 30 seconds.
[0057] Preferably, the biomass is washed with at most 6 volumes of
water per 1 volume of biomass, preferably with at most 3 volumes of
water.
[0058] Preferably, the biomass suspension is neutralized to pH 7 by
adding KOH or NaOH, preferably by adding KOH.
[0059] Preferably, the cells of the biomass are lyzed by milling,
preferably bead milling.
[0060] Preferably, the biomass is concentrated by centrifugation or
evaporation.
[0061] Optionally, the effects of the steps of processing the
microalgal biomass on the quality of the product may be determined
by one or more of the following parameters: [0062] measuring the
dry cell weight in the biomass; [0063] measuring the sugar content;
[0064] determining the amount of proteins; [0065] analyzing the
volatile organic compounds; [0066] measuring enzyme activities, in
particular lipoxygenase activity; [0067] measuring the coloration
or the pigment content; [0068] measuring the content of metals, in
particular iron, copper or nickel; [0069] determining the degree of
oxidation.
[0070] In particular, the effects of several biomass processing
operations on the quality of the product are compared and the
processing operation(s) giving the best results are selected.
[0071] In a particular embodiment, the microalgae of the Chlorella
genus are chosen from the group consisting of Chlorella vulgaris,
Chlorella sorokiniana and Chlorella protothecoides, and are more
particularly Chlorella protothecoides.
[0072] The aim of this method is, in particular, to develop an
optimized method for the production of protein-rich microalgal
biomass at high yield; said biomass having no undesirable flavors
or odors and being stable over time.
DETAILED DESCRIPTION OF THE INVENTION
[0073] The present invention relates to an optimized method which
allows all the requirements specific to the production of a
protein-rich microalgal biomass to be met, more particularly in
terms of protein production efficiency, in terms of organoleptic
quality and in terms of stability over time of said biomass.
[0074] The present invention thus relates to a method for
optimizing the downstream processing of a protein-rich biomass of
microalgae of the Chlorella genus which has been prepared
beforehand by fermentation in heterotrophic conditions and in the
absence of light, comprising: [0075] 1) providing a biomass
comprising more than 50% proteins by dry weight of biomass; [0076]
then, at low temperature: [0077] 2) recovering the biomass at the
end of fermentation, [0078] 3) washing and concentrating the
biomass, [0079] 4) optionally lyzing the biomass, then, with no low
temperature constraints: [0080] 5) optionally concentrating the
biomass suspension, [0081] 6) applying a heat treatment, [0082] 7)
drying the resulting biomass, to obtain the product, [0083] a step
of adjusting the pH to 7 being applied before or after step 6) of
heat treatment.
[0084] According to step 1) of the method in accordance with the
invention, the biomass comprises at least 50% by dry weight of
proteins. Even more preferably, it comprises at least 55, 60, 65 or
70% by dry weight of proteins.
[0085] The preferred microalgae of the invention can grow in
heterotrophic conditions (on sugars as source of carbon and in the
absence of light). The applicant company recommends choosing
protein-rich microalgae of the Chlorella genus. The microalgae used
may be chosen, nonexhaustively, from Chlorella protothecoides,
Chlorella kessleri, Chlorella minutissima, Chlorella sp., Chlorella
sorokiniana, Chlorella luteoviridis, Chlorella vulgaris, Chlorella
reisiglii, Chlorella ellipsoidea, Chlorella saccarophila,
Parachlorella kessleri, Parachlorella beijerinkii, Prototheca
stagnora and Prototheca moriformis. Preferably, the microalgae used
according to the invention belong to the species Chlorella
protothecoides.
[0086] In a very particular embodiment, the strain of Chlorella
sorokiniana is the strain UTEX 1663 from The Culture Collection of
Algae at the University of Texas at Austin, USA. In a very
particular embodiment, the strain of Chlorella protothecoides is
the strain CCAP211/8D from The Culture Collection of Algae and
Protozoa, Scotland, UK. The microalgae are cultured in liquid
medium to produce the biomass proper. According to the invention,
the microalgae are cultured in a medium containing a source of
carbon and a source of nitrogen in the absence of light
(heterotrophic conditions). The solid and liquid growth media are
generally available in the literature, and recommendations for the
preparation of particular media suitable for a wide variety of
strains of microorganisms may be found, for example, online at
www.utex.org/, a site run by the University of Texas at Austin for
its culture collection of algae (UTEX). The production of biomass
is carried out in fermentors (or bioreactors).
[0087] The specific examples of bioreactors, culture conditions and
heterotrophic growth and propagation methods may be combined in any
appropriate way to improve the efficiency of the microalgal growth
and the protein content. The production methods for such a biomass
are well known to those skilled in the art.
[0088] Steps 2 to 4 of the method in accordance with the invention
are carried out at low temperature, that is to say at a temperature
kept lower than 8.degree. C., preferably lower than 4.degree. C.
This low temperature enables cellular metabolism, and also the
development of microbial contaminants, to be stopped/slowed
down.
[0089] Moreover, as the applicant company has noted, another
advantage of carrying out these steps at low temperature is that
the cooling, and also the limited oxygenation, promote limitation
of the oxidative phenomena which cause "off notes" and which are a
source of instability in the final product.
[0090] More particularly: [0091] In step 2) of the method in
accordance with the invention, the biomass is recovered at the end
of the fermentation. [0092] Advantageously, the biomass is
recovered as soon as the residual source of nutrition (in
particular the residual glucose) is used up. [0093] These
conditions enable the production efficiency of the biomass produced
to be optimized, and the concentration of residual soluble matter
which has to be removed during the washing step to be limited.
[0094] In step 3) of the method in accordance with the invention,
the biomass is washed and concentrated. The biomass is washed of
the residual soluble matter at the end of fermentation (salts,
non-metabolized sugars, etc.), by dilution with water. [0095] The
biomass is washed with at most 6 volumes of water per volume of
biomass, preferably with at most 3 volumes of water per volume of
biomass, and in a very particular embodiment with around one volume
of water per volume of biomass. [0096] This operation makes it
possible to significantly improve the cell purity (reduce the
fraction of solids at the end of fermentation which is derived from
a non-cellular component). [0097] In this way, the load of this
soluble matter, which is potentially a source of degradation of the
sensory properties of the biomass, is reduced. [0098] This
operation is advantageously carried out in low temperature
conditions. [0099] The biomass is then concentrated to 15 to 40%
solids, preferably 20-30% solids. [0100] It may be concentrated by
centrifugation, for example using an Alfa Laval FEUX 510
centrifuge. [0101] In step 4), the resulting biomass is optionally
lyzed. [0102] The cell walls and the intracellular components are
milled or reduced. [0103] Various techniques are available for
carrying out the lysis, such as microbead milling and high-pressure
homogenization technology. [0104] The preferred mode is microbead
milling, in particular microbead milling using a Bead Mill. [0105]
Conventionally, a NETZSCH Labstar bead mill is used with zirconium
silicate beads of 0.5 mm diameter.
[0106] The degree of lysis may be variable. For example, a degree
of lysis of 50, 60, 70, 80, 90 or 95% of the cells may be
envisioned.
[0107] The final three steps of the method in accordance with the
invention are carried out with no low temperature constraints.
[0108] In step 5), the resulting biomass suspension is optionally
concentrated. [0109] The biomass is concentrated by evaporation.
Any type of evaporator may be used, for example a rotary
evaporator, a forced flow evaporator, a falling film evaporator or
a wiped film evaporator. [0110] Concentration by evaporation
contributes to improving the concentration factor before drying by
optimizing the energy performance of the method. This concentration
also makes it possible to strip out any volatile products which are
potentially deleterious to the sensory properties of the final
product. [0111] In step 6), a heat treatment is applied. [0112]
This heat treatment acts as a safety measure to counter any
microbiological risks to the final product. [0113] Conventionally
it consists of an HTST or UHT treatment. Moreover, this heat
treatment contributes to improving the sensory properties of the
final product. [0114] Two different types of heat treatment are
envisioned in particular. [0115] The first type is a high
temperature/short time (HTST) heat treatment of the biomass, for
example for 30 seconds to 5 minutes at a temperature lower than
100.degree. C. [0116] The second type is a UHT (ultra-high
temperature) heat treatment. Preferably, the UHT heat treatment is
carried out at a temperature of between 100 and 150.degree. C. for
5 to 30 seconds, preferably at a temperature of between 120 and
140.degree. C. for 5 to 15 seconds. [0117] In step 7), the
resulting biomass is dried to obtain the product. [0118]
Preferably, the drying is carried out by spray drying. [0119] Spray
drying is carried out in a spray dryer in which a liquid suspension
is sprayed, in the form of a dispersion of fine droplets, into a
stream of heated air, with the material carried along being rapidly
dried and forming a dry powder. T [0120] here are many devices in
the prior art for spray drying lipid-rich compounds. It is possible
to readily find in the literature illustrations of the equipment
and technology proposed: for example, in the Spray Drying Handbook
by K. Masters, in particular in the 5.sup.th edition thereof,
published in 1991 and republished in 1994 by Longman Scientific
& Technical (available at the British Library or the Library of
Congress under ISBN 0-470-21743-X), or in the BETE.RTM. Spray Dry
Manual, 2005 (accessed at the website www.bete.com). [0121] For
example, the spray drying may be carried out on a Niro Mobile Minor
single-effect spray drying tower or on a Filtermat FMD125 with
cyclone.
[0122] A final, key step consists in neutralizing the pH of the
(lyzed or not lyzed) biomass suspension to 7 before or after step
6) of heat treatment.
[0123] This neutralization may be carried out by adjusting the pH
to 7 by adding NaOH or KOH, preferably KOH. This neutralization
with concentrated potassium hydroxide makes it possible to smooth
out any possible fluctuations in pH downstream and between
production batches, and also to improve the sensory properties.
[0124] The addition of one or more antioxidants may also be chosen
(before or after the step of neutralizing the pH to 7).
[0125] It is possible, advantageously, to choose ascorbic acid
and/or a mixture of tocopherols, preferably a combination of
ascorbic acid and tocopherols.
[0126] Conventionally, the proportions used are 150 ppm/dry of
ascorbic acid and 500 ppm/dry of a mixture of tocopherols
(.times.ppm/dry meaning.times.mg per kg of dry biomass).
[0127] It is clearly understood that the nature of the antioxidant
depends on the properties of the matrix to be stabilized. They must
improve the stability of the final product with regard to the risks
of oxidative modification, and thereby improve the preservation of
the final product by retaining a stable physicochemical and sensory
profile.
[0128] The effects of the steps of processing the microalgal
biomass on the quality of the product may moreover be determined
by: [0129] measuring the loss of yield, in particular analyzing the
loss of cellular solids and also the loss in protein content
arising in particular from "dissolution" during the heat treatment
and the elimination thereof upon washing, if the latter step is
carried out downstream. [0130] It has been shown that this loss of
solids predominantly consists of a protein fraction (which allows
the difficulties a) and b) identified above to be addressed);
[0131] determining the sensory quality of the batches produced, in
particular by a sensory panel formed to evaluate the sensory
properties of various batches (to address difficulty c)); [0132]
measuring stability over time, in particular by an accelerated
aging test consisting of comparative sensory analysis of the
initial sample and of this same sample placed in an oven under
hermetic conditions for 10 days at 60.degree. C. (to address
difficulty d)). [0133] The sensory analysis is carried out in
accordance with the test described. This analysis makes it possible
to highlight any oxidation descriptors, thereby making it possible
to evaluate the stability of the sample with regard to this
oxidative degradation; [0134] measuring the change in microbial
load over time on the various steps of the method (to address
difficulty e)); [0135] analyzing the change in the solids
(concentration factor) over this series of operations (to address
difficulty f)).
[0136] Three characteristics essential to evaluating the quality
have been defined by the applicant company: [0137] the content of
dry biomass and/or proteins in the product; [0138] the organoleptic
quality of the product; and [0139] the stability over time of the
product.
[0140] Moreover, other parameters may also be taken into account
for evaluating the quality of the product, in particular the
following: [0141] measuring the dry cell weight in the biomass;
[0142] measuring the sugar content; [0143] determining the amount
of proteins; [0144] analyzing the volatile organic compounds;
[0145] measuring enzyme activities, in particular lipoxygenase
activity; [0146] measuring the coloration or the pigment content;
[0147] measuring the content of metals, in particular iron, copper
or nickel; [0148] determining the degree of oxidation.
[0149] The invention will be better understood by virtue of the
following examples which are intended to be non-limiting and
illustrative.
EXAMPLES
[0150] Several batches were produced by downstream processing of a
biomass of Chlorella protothecoides prepared by fermentation in
heterotrophic conditions and in the absence of light. The strain
used is Chlorella protothecoides with the reference UTEX 250.
[0151] The various steps were carried out as defined below.
[0152] HTST treatment: high temperature/short time (HTST) heat
treatment of the biomass, for 30 seconds to 5 min, at a temperature
lower than 100.degree. C., in particular for 1 minute at 75.degree.
C.
[0153] Washing: with at most 6 volumes of water per volume of
biomass.
[0154] Addition of antioxidant: addition of ascorbic acid and of a
mixture of tocopherols, preferably with proportions of 150 ppm/dry
of ascorbic acid and 500 ppm/dry of a mixture of tocopherols.
[0155] Spray drying: spray drying on a Niro Mobile Minor
single-effect spray drying tower or on a Filtermat FMD125 with
cyclone.
[0156] Milling: bead milling using a Bead Mill. Conventionally, a
NETZSCH Labstar bead mill is used with zirconium silicate beads of
0.5 mm diameter.
[0157] Concentrating: concentration/evaporation by rotary
evaporator (laboratory scale) or any other type of larger-scale
evaporator (forced flow, falling film, wiped film, etc.) of 20 to
30% solids.
[0158] UHT treatment: at a temperature of between 100 and
150.degree. C. for 5 to 30 seconds, preferably at a temperature of
between 120 and 140.degree. C. for 5 to 15 seconds.
[0159] The quality of the batches obtained is studied in the
following way. One or more of the following parameters were
determined or measured.
[0160] Sensory quality: the sensory quality of the batches produced
is evaluated by a sensory panel of approximately 18 people for a
set of sensory descriptors. The expert panel evaluates the
olfactory properties of the batches at 3% in water and at
55.degree. C. (samples presented in a closed glass jar), on ordinal
intensity scales (NF V 09-015:1985).
[0161] Stability over time: this is measured during an accelerated
aging test developed by the applicant company which consists of a
comparative sensory analysis of the initial sample and of this same
sample placed in an oven under hermetic conditions for 10 days at
60.degree. C. The sensory analysis makes it possible to highlight
any oxidation descriptors, thereby making it possible to evaluate
the stability of the sample with regard to this oxidative
degradation.
[0162] Measuring the loss of yield: [0163] measuring the dry cell
weight (DCW) and/or [0164] measuring the dry biomass and/or [0165]
the protein content.
[0166] Moreover, additional parameters may also be evaluated.
[0167] Sugar content: determining the sugar (glucose, maltose,
fructose, sucrose) content by liquid chromatography. Following
separation by ion exchange chromatography, the various species are
detected by amperometric analysis.
[0168] Volatile organic compounds: the content of volatile organic
compounds is determined by SPME/GC.
[0169] Analysis of the heat treatment: by observing, by optical
microscopy, changes brought about in cell morphology.
[0170] Measuring the coloration: measuring, by means of a
spectrocolorimeter, reflectance measurements at wavelengths from
400 nm to 700 nm under the D65 or C illuminant and with the CIE
1931 2.degree. observer. The indices "L", "a" and "b" are
determined, where "L" corresponds to lightness, "a" to the green to
red scale and "b" to the blue to yellow scale.
[0171] Pigment content: after breaking the cells open, the pigments
are extracted with 90% acetone. The extract is then analyzed by
spectrophotometry. The pigments are quantified by calculations
based on the absorbances recorded at various wavelengths.
[0172] Assaying of metals: destruction of the organic material by
mineralization using a sulfonitric mixture and subsequent
determination by emission spectrometry following appropriate
dilution.
[0173] Determining the degree of oxidation: after dilution in
isooctane, measurement of the absorbance at 232 nm.
* * * * *
References