WO2015063282A1 - Utilisation d'algues pour accroître la biomasse active viable de bactéries d'acide lactique - Google Patents

Utilisation d'algues pour accroître la biomasse active viable de bactéries d'acide lactique Download PDF

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Publication number
WO2015063282A1
WO2015063282A1 PCT/EP2014/073505 EP2014073505W WO2015063282A1 WO 2015063282 A1 WO2015063282 A1 WO 2015063282A1 EP 2014073505 W EP2014073505 W EP 2014073505W WO 2015063282 A1 WO2015063282 A1 WO 2015063282A1
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algae
lab
fraction
culture
lactic acid
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PCT/EP2014/073505
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English (en)
Inventor
Michel CADENEL
Jean-Philippe Obert
Isabelle Auzanneau
Frédéric LOMBARDI
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Dupont Nutrition Biosciences Aps
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Publication of WO2015063282A1 publication Critical patent/WO2015063282A1/fr

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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING THEREOF
    • A23C9/00—Milk preparations; Milk powder or milk powder preparations
    • A23C9/12—Fermented milk preparations; Treatment using microorganisms or enzymes
    • A23C9/123—Fermented milk preparations; Treatment using microorganisms or enzymes using only microorganisms of the genus lactobacteriaceae; Yoghurt
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/12—Unicellular algae; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/38—Chemical stimulation of growth or activity by addition of chemical compounds which are not essential growth factors; Stimulation of growth by removal of a chemical compound

Definitions

  • the present invention relates to the field of bacterial culture, particularly the culture of lactic acid bacteria, and most particularly the culture of lactic acid bacteria under aerobic growth conditions and the use of algae to increase the viable active biomass of lactic acid bacteria under aerobic growth conditions, as well as LAB compositions and harvested LAB compositions produced in this way.
  • Lactic acid bacteria are extensively cultured worldwide for use in the food and feed industry. They are used in the manufacture of fermented products including dairy, meat, bakery, wine and vegetal products. Consumption of fermented products has lately been increasing worldwide, and therefore there is a need to find ways to increase the active biomass of LAB produced by culture.
  • LAB biomass production can be achieved following two different metabolic pathways: aerobic and anaerobic. It has been described that in LAB, aerobic respiration is activated by exogenous heme or haem (Lechardeur et ah Current Opinion in Biotechnology 201 1 , 22:143-149). Hemin is an iron containing porphyrin compound, derived from red blood cells. In optimal aerobic conditions, the LAB cells are able to use oxygen to produce ATP with a higher efficiency that results in a higher biomass yield and a lower production of inhibitory lactate compared to anaerobic fermentation. Therefore additives such as hemin, which increase aerobic respiration and thus biomass of LAB in culture, are valuable in industry. Hemin for use in LAB culture has usually been sourced from porcine or bovine hemoglobin.
  • the Inventors have identified other cell growth activators, algae or fractions thereof, which can be used to increase growth of LAB under aerobic conditions.
  • Algae are photosynthetic organisms both unicellular and multicellular. Algae are mostly eukaryotic organisms and can be classified based on their pigmentation, and thus encompass red algae (Rhodophyta), brown algae (Heteromontophyta), green algae l (Chlorophyta) and Diatomaceae.
  • the term "algae” also encompasses the prokaryotic algae, such as cyanobacteria informally referred to as blue-green algae.
  • Algae and extracts thereof are known to increase the growth of some LAB under anaerobic conditions (see for example Bhowmik et al. World Journal of Dairy & Food Sciences (2009), 4(2), pp160-163). Algae extracts are also known to influence the production of acid in milk under anaerobic conditions, which is known as the booster effect of algae on LAB (Molnar et al. Milchwissenschaft (2005), 60(4), pp380-382). However, the specific uses, methods and culture conditions of the inventions disclosed herein have not previously been described.
  • the present invention provides the use of an algae of a fraction thereof, in particular an algae selected from the group consisting of spirulina, chlorella and a fraction thereof, to increase under aerobic growth conditions the biomass, in particular the viable active biomass, of lactic acid bacteria (LAB). Furthermore, a method of producing a LAB composition and harvested LAB composition with increased viable active biomass is provided.
  • the present invention provides an increased biomass, in particular an increased viable and/or active biomass, of LAB in aerobic culture, through the use of algae and fractions thereof.
  • the present invention details the use of an algae or a fraction thereof selected from the group consisting of spirulina, chlorella and a fraction thereof, to increase under aerobic growth conditions the biomass, in particular the viable active biomass, of LAB.
  • the present invention uses specific assays to identify and quantify the increase in biomass of LAB.
  • the present invention also provides LAB compositions and harvested LAB compositions, including intermediate LAB starter cultures, LAB starter cultures and probiotics, particularly intermediate LAB starter cultures and LAB starter cultures for use in producing fermented products.
  • the use of algae or a fraction thereof in culturing LAB increases the biomass, in particular the viable active biomass, of LAB produced.
  • the methods and uses of the present invention reduce cost associated with the production of LAB compositions and harvested LAB compositions.
  • the LAB compositions and harvested LAB compositions produced by the methods of the invention have a reduced lag phase (Ta value) and are therefore faster acidifiers.
  • the current invention provides a method of increasing LAB biomass as demonstrated by a reduced lag phase (Ta value) and/or measurable increase in the colony forming units (CFU) of the LAB composition or harvested LAB composition of the invention.
  • the current invention provides at least an equivalent, or even an increased, LAB viable active biomass compared to the use of hemin instead of algae or a fraction thereof.
  • the current invention further provides a LAB composition, a harvested LAB composition, intermediate starter culture, starter culture or LAB probiotic composition.
  • Algae are a vegetal additive. Therefore the use of algae or a fraction thereof, instead of animal-derived hemin, allows consumers with diets such as vegetarian, vegan, kosher and Halal to consume the resulting products.
  • algae or a fraction thereof is easily produced and found worldwide, and therefore can be sourced relatively cheaply and easily.
  • Figure 1 Spirulina concentration impact on Ta activity (minutes) of Lactococcus lactis DGCC2631 cultured under aerobic conditions
  • Figure 3 Spirulina concentration impact on CFU concentration (CFU/ml) of Lactococcus lactis DGCC2631 cultured under aerobic conditions
  • Figure 4 Chlorella concentration impact on CFU concentration (CFU/ml) of Lactococcus lactis DGCC2631 cultured under aerobic conditions
  • Figure 5 Spirulina concentration impact on OD gain (delta OD) of Lactococcus lactis DGCC2631 cultured under aerobic conditions
  • the present inventors have surprisingly found that some algae or a fraction thereof can be used to increase the biomass, in particular the viable active biomass, of lactic acid bacteria (LAB), under aerobic growth conditions.
  • LAB lactic acid bacteria
  • the invention is directed to a method of producing a LAB composition with increased LAB viable active biomass comprising the steps of:
  • said increased viable active biomass is defined as at least one of the following: a) the lag phase of growth on a fermentable substrate (Ta value), of the lactic acid bacteria composition, or a sample thereof, is at least 10 minutes less than the Ta value of a LAB culture medium, or a same sized sample thereof, obtained after culture in the absence of said algae or a fraction thereof; and/or
  • the content of viable cells, expressed in colony forming units (CFU), of the lactic acid bacteria composition, or a sample thereof is at least 120% the content of viable cells of a LAB culture medium, or a same sized sample thereof, obtained after culture in the absence of said algae or a fraction thereof.
  • CFU colony forming units
  • the invention is also directed to a method of producing a harvested LAB composition comprising the steps of: (i) admixing a culture medium and a LAB, and an algae or a fraction thereof; (ii) culturing said LAB admixture under aerobic growth conditions to produce a LAB composition with increased lactic acid bacteria viable active biomass; wherein said increased viable active biomass of the LAB composition is defined as at least one of the following: a) the lag phase of growth on a fermentable substrate (Ta value), of the lactic acid bacteria composition, or a sample thereof, is at least 10 minutes less than the Ta value of a LAB culture medium, or a same sized sample thereof, obtained after culture in the absence of said algae or a fraction thereof; and/or
  • the content of viable cells, expressed in colony forming units (CFU), of the lactic acid bacteria composition, or a sample thereof, is at least 120% the content of viable cells of a LAB culture medium, or a same sized sample thereof, obtained after culture in the absence of said algae or a fraction thereof;
  • step iii) harvesting the LAB from the LAB composition of step ii) by separating the LAB from the final culture medium, such as at least 80% of the final culture medium is removed and/or by separating the LAB from a portion of the algae or a fraction thereof such that at least 5% of the algae or a fraction thereof initially admixed in step (i) is removed, to produce a harvested LAB composition.
  • the harvesting step is separating the LAB from the final culture medium, such as at least 80% of the final culture medium is removed In a particular embodiment, the harvesting step is separating the LAB from a portion of the algae or a fraction thereof such that at least 5% of the algae or a fraction thereof initially admixed in step (i) is removed.
  • the harvested LAB composition when at least 80% of the final culture medium is removed, also has an increased lactic acid bacteria viable active biomass, wherein said increased viable active biomass of the harvested LAB composition is defined as at least one of the following: a) the lag phase of growth on a fermentable substrate (Ta value), of the harvested lactic acid bacteria composition, or a sample thereof, is at least 10 minutes less than the Ta value of a harvested LAB culture medium, or a same sized sample thereof, obtained after culture in the absence of said algae or a fraction thereof; and/or b) the content of viable cells, expressed in colony forming units (CFU), of the harvested lactic acid bacteria composition, or a sample thereof, is at least 120% the content of viable cells of a harvested LAB culture medium, or a same sized sample thereof, obtained after culture in the absence of said algae or a fraction thereof
  • the invention also provides for the use of an algae or a fraction thereof, in the manufacture of a lactic acid bacteria composition for increasing viable active biomass of LAB, cultured under aerobic growth conditions, wherein increased viable active biomass is defined as at least one of the following: a) wherein the lag phase of growth on a fermentable substrate (Ta value) of the lactic acid bacteria composition, or a sample thereof, is at least 10 minutes less than the Ta value of a LAB culture medium, or same sized sample thereof, obtained after culture in the absence of said algae or a fraction thereof; and/or b) wherein the content of viable cells, expressed in colony forming units (CFU), of the lactic acid bacteria composition, or a sample thereof, is at least 120% the content of viable cells of a LAB culture medium, or a same sized sample thereof, obtained after culture in the absence of said algae or a fraction thereof.
  • Ta value lag phase of growth on a fermentable substrate
  • CFU colony forming units
  • the invention also provides for the use of an algae or a fraction thereof, in the manufacture of a harvested lactic acid bacteria composition for increasing viable active biomass of LAB, cultured under aerobic growth conditions, wherein increased viable active biomass is defined as at least one of the following:
  • the lag phase of growth on a fermentable substrate (Ta value) of the harvested lactic acid bacteria composition, or a sample thereof is at least 10 minutes less than the Ta value of a harvested LAB culture medium, or same sized sample thereof, obtained after culture in the absence of said algae or a fraction thereof;
  • a “fermentable substrate” is a material that contains an organic compound such as a carbohydrate that can be transformed (i.e., converted into another compound) by the enzymatic action of a bacterium such as LAB.
  • substrate also means a fermentable substrate.
  • a fermentable substrate may consist of, contain or comprise milk, non-fat dry milk, vegetables (e.g., corn potatoes, cabbage), starch, grains (e.g. rice, wheat, barley, hops), fruit (e.g., grapes, apples, oranges), sugar, sugarcane, meat (e.g., beef, poultry, pork, sausage), heart infusion, cultured dextrose, combinations thereof, and media containing proteins, carbohydrates, and minerals necessary for optimal growth.
  • the fermentable substrate is milk, which may be referred to as "milk substrate”.
  • active in the context of the present invention means lactic acid bacteria, and/or a culture or composition thereof, which are able to produce lactic acid after incubation or culture in a fermentable substrate (suitably milk), in order to decrease the pH.
  • a fermentable substrate suitable for milk
  • the pH decrease is at least 0.1 and at most 3 pH units.
  • viable in the context of the invention means lactic acid bacteria which are able to grow and to multiply on an appropriate medium, for example a complex medium, a specified medium, an enriched medium, nutrient medium and/or Medium A of the examples.
  • the medium may be liquid, liquid paste or solid, such as agar medium.
  • “viable” refers to LAB which contain cytoplasmic enzyme activity, identified by specific fluorochromes and/or by appropriate staining and/or which present cell membrane integrity, for example identified by direct epifluorescent filter technique (DEFT - see for example Bunthof CJ, Bloemen K, Breeuwer P, Rombouts
  • Viable LAB may also be referred to as "viable cells".
  • the aim of the use or method of the invention is to increase the viable active biomass of LAB and/or to produce a LAB composition and/or a harvested LAB composition with increased LAB viable active biomass, wherein "active" and “viable” are defined as above.
  • medium means a composition, which may be liquid, gelatinous or solid, containing nutrients, which may include carbon, nitrogen, minerals, vitamins and/or fat sources, preferably at an optimum concentration, in which LAB can grow and multiply.
  • the culture medium is not milk.
  • the culture medium is not skim milk, skimmed milk, milk-containing medium, skim milk- containing medium, skimmed milk-containing medium, reduced fat milk or 0.1 % milk.
  • a "LAB culture medium” is separate and is defined in more detail below.
  • the medium may comprise a carbohydrate, a yeast or yeast extract, buffer, surfactant, vitamin and/or nutrient sources (such as a vitamin C source), defoamer (or antifoamer, or anti-foamer) and a solvent (suitably water).
  • the medium may comprise lactose, a yeast extract, magnesium sulfate heptahydrate (MgS0 4 7H 2 0), Manganese(ll) sulfate monohydrate (MnS0 4 , H 2 0), Na ascorbate, defoamer and soft water (water with a reduced content of cations and anions).
  • the culture medium is the medium A as defined in point 1 .3 of the examples below.
  • a medium may also be referred to as a "nutrient medium”, “nutrient media”, “growth media” or “nutrient and growth media/medium”.
  • the medium is not MRS.
  • culturing as used herein means any method to grow and to multiply microbial organisms, such as LAB, by enabling them to reproduce in predetermined culture medium under controlled conditions of pH, temperature oxygen content, stirring and length of growing time (also known as culture length).
  • culture may be used to refer to the culturing of any microbial organisms, such as one or more LAB strains as exemplified below.
  • culturing is aerobic or under aerobic growth conditions as defined below.
  • admixing as used herein means mixing together to combine or form one substance.
  • the term "admixing” refers to mixing a culture medium, lactic acid bacteria and an algae or a fraction thereof.
  • the mixing may be carried out in any order.
  • the culture medium may be mixed with the algae or a fraction thereof prior to mixing this combination with a lactic acid bacteria.
  • the combination of the algae or a fraction thereof and the culture medium may be optionally sterilised prior to adding the lactic acid bacteria.
  • Components which have bee admixed may be refereed to herein as an admixture or mixture.
  • anobic growth conditions refers to growth requiring the presence of oxygen, preferably dissolved oxygen, in the culture medium or the LAB culture medium.
  • oxygen preferably dissolved oxygen
  • Aerobic growth conditions may be achieved by continuous aeration, e.g. continuous air flow and/or agitation (e.g. stirring, rotating and/or shaking).
  • continuous aeration e.g. continuous air flow and/or agitation (e.g. stirring, rotating and/or shaking).
  • Aerobic growth conditions may for example be obtained by continuous agitation, such as rotating and/or stirring and/or shaking, at normal air atmosphere (e.g. 1 atm).
  • agitation e.g. stirring, rotating and/or shaking
  • 10- l OOOrpm 100-500rpm, 100-300rpm, 100-200 rpm or 100-200 rpm.
  • agitation occurs at 160rpm.
  • agitation occurs at 30-150rpm, preferably 50-100rpm.
  • aerobic growth conditions can be achieved by a continuous air flow, for example by using bubbling, or by air circulation such as by using an airlift such as in an air lift-type fermentor.
  • Dissolved Oxygen (03 ⁇ 4 is preferably controlled at a value of 10-30%, suitably at a value of 15-25% and most preferably at around 20% or 20%.
  • Dissolved 0 2 may be maintained at the required value for example by using a probe for monitoring, such as using a p02 probe for monitoring.
  • the dissolved 0 2 in the culture media may be controlled by the regulation of the air injection flow rate, such as by using a air sparger or microsparger, and/or by bubbling air at the bottom of the fermentor wherein the LAB is cultured.
  • an air flow rate from 0.3vvm-5vvm, suitably 1 -4vvm, suitably 2-3vvm may be used.
  • Aeration may be carried out by continuous rotating and/or shaking and/or stirring.
  • the energy an impeller or stirring device dissipates into the liquid/broth is in the range of 0.2 to 5 kW/m3, in particular 1 to 2 kW/m3.
  • the culturing is carried out in a fermentor, e.g. a fermenting vat.
  • the culturing is carried out in an Erlenmeyer flask.
  • large fermentors (industrial scale) are used, with an air flow rate at 0.3vvm to 5vvm, and the culture is stirred with an energy dissipation of the impeller between 0.2 to 5 kW/m3, in particular 1 to 2 kW/m3, to produce aerobic growth conditions.
  • a 2L fermentor with an air flow rate at 0.3vvm to 5vvm, and stirring from 500 to 1000 rpm may be used.
  • shaking may occur at 160rpm. This may be considered equivalent to 0.3vvm to 5vvm using an impeller for stirring, such as a Rushton impeller type, stirring from 200 rpm to maximum of l OOOrpm.
  • Foam formation in the medium may be controlled by the admixing, preferably injection of, appropriate defoamer or anti-foamer. Preferably such injection may be carried out when a conductivity sensor is in contact with the foam. Foam formation may also be controlled using a mechanical foam breaker in one embodiment.
  • culturing the LAB is carried out in a controlled pH environment. Suitably the pH of the medium is therefore controlled at or around 5-7, most suitably pH 6-7 and in one embodiment pH is controlled at 6.2. In one embodiment, culturing the LAB (in the method or use of the invention) is carried out at a controlled temperature.
  • the temperature is from 10-50 S C, most suitably 20-45 s C, most suitably the temperature is 25-35 S C and more suitably culturing is carried out at a temperature of 30 S C or at around 30 S C, such as 26, 27, 28, 29, 30, 31 , 32, 33 or 34 e C .
  • Culturing is carried out for at least 1 -30 hours, most suitably 4-24 hours, more suitably 10-20 hours, more suitably 15-20 hours, more suitably 16-18 hours and most suitably for at or around 18 hours.
  • the culture is stopped when the LAB in the LAB composition have reached a sufficiently high concentration, in particular have reached at least a concentration of 10 8 to 10 2 CFU / ml of the LAB composition, most suitably at least 10 9 , at least 10 10 , at least 10 11 , at least 10 12 CFU / ml of the LAB composition.
  • the increase in LAB in the LAB composition during culturing is between 10,000 to 10,000,000 fold. Most preferably the increase in LAB during culturing is between 10,000 to 1 ,000,000 fold.
  • the LAB in the LAB composition increases (e.g. from about 10 4 CFU / ml of culture medium at the start of culturing) to 10 8 to 10 12 CFU / ml of the LAB composition (at the end of the culturing).
  • the LAB in the LAB composition increases to at least 10 9 CFU / ml of the LAB composition (at the end of the culturing), e.g. at least 10 10 , at least 10 11 CFU / ml of the LAB composition.
  • aerobic growth conditions are as follows: in a 1 L Erlenmeyer flask, at least 100 ml of culture medium, comprising the algae or fraction thereof at the desired concentration and admixed with a selected LAB, incubated for 18h at 30 °C on a shaker at 160 rpm (for example an INFORS shaker).
  • the uses and methods of the invention comprise culturing at least one strain of lactic acid bacteria under aerobic conditions in an appropriate nutrient and growth medium, at an appropriate temperature, in which at least one algae or a fraction thereof is present or is added.
  • the parameter "7a" is the time in minutes for the LAB composition or harvested LAB composition to decrease the pH of a fermentable substrate by 0.08 pH units.
  • the fermentable substrate is milk.
  • Ta is the time taken for the lag phase of growth.
  • the Ta value is determined after the initial culture of LAB under aerobic growth conditions. In other words, a sample or starter culture obtained from the LAB composition (from step (ii) of the method) is grown on a fermentable substrate in order to determine the Ta value.
  • Ta with algae or “Ta algae” may be used to indicate the Ta of a LAB composition or harvested LAB composition or a sample thereof, cultured under aerobic growth conditions in the presence of algae or a fraction thereof.
  • "Ta control” is the Ta of the LAB culture medium or the harvested LAB culture medium, which has been cultured under identical aerobic growth conditions, and harvested by identical harvesting methods if the LAB composition under the determine "Ta algae” was a harvested LAB composition.
  • it is the Ta of an identical culture of LAB or same sized sample thereof, wherein the algae or a fraction thereof has been excluded from the culturing process. The only difference is that the control does not include algae or a fraction thereof in the culture.
  • Ta with algae minus Ta control is the difference in minutes between the Ta of a LAB composition or harvested LAB composition or a sample thereof (after culture under aerobic conditions in presence of the algae or a fraction thereof) and the Ta of the LAB culture medium or the harvested LAB culture medium (which is the same LAB composition or same harvested LAB composition or same sized sample thereof after culture under the same aerobic conditions in absence of the algae or a fraction thereof) which is the control.
  • a reduction in the Ta value may be symbolised by a minus value. For example "-10" indicates a reduction in Ta of 10 minutes.
  • the Ta value is determined using Assay I as described herein.
  • the difference between the Ta algae and the Ta control of at least 10 minutes means that the addition of the algae or a fraction thereof produced a LAB composition or harvested LAB composition after culture which decreases the lag phase in milk, i.e., that the LAB of the composition after culture in presence of algae or a fraction thereof are more active in milk.
  • the Ta value difference between the Ta algae and the Ta control is at least 10 minutes, at least 12 minutes, at least 15 minutes, at least 20 minutes, at least 30 minutes or at least 40 minutes.
  • the results achieved by the present invention are not due to the addition of algae or a fraction thereof to the fermentable substrate (e.g. milk).
  • the LAB in the composition or harvested LAB composition of the present invention are more active (as measured by Ta decrease in milk) than identical LAB cultured in identical conditions without algae or a fraction thereof (see example 2.3).
  • the effects achieved with the present invention are over and beyond those which would be achieved by addition of algae (or a fraction thereof) alone to the milk.
  • the effect of the algae (or a fraction thereof) on the LAB in the LAB composition or harvested LAB composition of the invention is to increase activity compared to addition of algae (or a fraction thereof) alone to milk, and therefore are not solely due to known booster effect.
  • the LAB composition is a harvested LAB composition, to determine the calculation "Ta with algae minus Ta control" as described above, it should be compared to a LAB culture medium or a same sized sample thereof (obtained after culture in absence of the algae (or a fraction thereof) and used as a control) which has undergone the same harvesting methods.
  • the parameter "CFU/ml” qualifies the concentration of viable biomass obtained in the LAB composition or harvested LAB composition and is expressed as CFU/ml (colony forming units) after enumeration on appropriate media such as agar.
  • CFU/ml colony forming units
  • the calculation "ratio CFU with algae / CFU control” is the ratio (expressed in %) of the CFU of the LAB composition or harvested LAB composition of the invention compared to the LAB culture medium or harvested LAB culture medium or a same sized sample thereof (obtained after culture in absence of the algae (or a fraction thereof) -control).
  • the LAB composition is a harvested LAB composition, to determine the ratio "CFU with algae / CFU control" as described above, it should be compared to a LAB culture medium or a same sized sample thereof (obtained after culture in absence of the algae (or a fraction thereof) and used as a CFU control) which has undergone the same harvesting methods.
  • CFU is determined using Assay II as described herein.
  • a ratio "CFU with algae / CFU control" of at least 120% means that the addition of the algae (or a fraction thereof) produced a LAB composition or harvested LAB composition after culture which contains more viable cells.
  • the ratio "CFU with algae / CFU control" is of at least 130, at least 140, at least 150 or at least 200 %, i.e., the content of viable cells of a lactic acid bacteria composition obtained after culture in the presence of said algae (or a fraction thereof) under aerobic conditions, is at least 130, at least 140, at least 150 or at least 200 % the content of viable cells of a lactic acid bacteria culture medium or a harvested lactic acid bacteria culture medium obtained after culture in the absence of said algae (or a fraction thereof).
  • algae refers to any algae including the prokaryotic algae and the eukaryotic algae, and encompasses also any fraction of algae, as defined in this application.
  • the algae used in the invention is a prokaryotic algae, in particular a cyanobacteria, or a fraction thereof.
  • said cynaobacteria is Spirulina, also known as Arthrospira (Arthrospira species,), such as Arthrospira platensis (or Spirulina platensis,) or Arthrospira maxima.
  • Spirulina may also be referred to as "Spiruline”.
  • the algae (or a fraction thereof) used in the invention is an eukaryotic algae, in particular a red algae, or a green algae or a fraction thereof.
  • the algae used in the invention is an eukaryotic algae selected from the red algae or the green algae.
  • the algae used in the invention is a green algae, in particular an algae from the Chlorophyta division, in particular from the Chlorellales order, in particular from the Chlorellaceae family, and in particular is a Chlorella species.
  • Chlorella encompasses species such as Chlorella vulgaris. It is well known as a health food (see for Example Becker, E.W. Biotechnol Adv. 2007 Mar-Apr;25(2):207-10).
  • the algae used in the invention are red algae, in particular an algae from the Rhodophyta division.
  • the algae when the algae (or a fraction thereof) is an eukaryotic algae or is selected from an eukaryotic algae and a prokaryotic algae, such as a cyanobacteria, said algae is not a brown algae, in particular is not an algae from the Phaeophyceae class.
  • the algae is not an algae from the Laminariales Order, in particular is not an algae from the Laminariaceae Family, in particular is not a Laminaria species (laminaire), such as Laminaria digitata.
  • the algae is not an algae from the Fucales order, in particular is not an algae from the Fucaceae family, in particular is not a Fucus species, such as Fucus vesiculosus.
  • the algae (or a fraction thereof) is a prokaryotic algae, in particular a cyanobacteria, or eukaryotic algae selected from the group consisting of a red algae and a green algae, or any fraction thereof.
  • the algae (or a fraction thereof) used in the invention are selected from the group consisting of Spirulina, Chlorella or a fraction thereof.
  • the algae used in the invention may include any fraction of said algae.
  • a fraction thereof when applied to algae refers to an extract, suspension, dilution, part, fragment of algae, such as spirulina fraction or chlorella fraction.
  • the fraction may be in dried, powdered, liquid, diluted, reconstituted powder suspension or other reconstituted form. It may also be a fresh extraction from growing algae such as growing spirulina or chlorella.
  • the fraction may include all active parts of the algae.
  • the fraction of algae is in the form of whole algae, powdered, and in a further embodiment the fraction of algae is in the form of whole algae, dried and powdered.
  • the term “dried” preferably means less than or equal to 8% humidity.
  • the term “powdered” preferably refers to a particle size of less 300 ⁇ mean diameter, preferably less than 250, 200, 190, 180, 170, 160, 150 140, 130, 120, 100, 90 or 80 ⁇ mean diameter.
  • the algae or fraction thereof is reconstituted in a liquid before use, preferably said liquid is water.
  • the algae may be commercially sourced.
  • An “extract” may refer to a liquid, aqueous, powdered or dried extract of any part of the algae.
  • a “suspension” may refer to any part or extraction of algae suspended in a liquid.
  • a “dilution” may refer to any part or any extraction, such as a liquid extraction, diluted with any liquid.
  • a “part” of an algae may refer to any section of algae including green parts, blade stipe, stem or holdfast.
  • Non limiting examples of spirulina fractions which may be used in the invention include, but is not limited to, Spirulina extract from the following providers: Spirulina from Setalg, Spirulina from SEAH, Spirulina from COPCI or Spirulina from AlphaBiotec Miette.
  • a non limiting example which may be used in the invention includes chlorella extract from Setalg.
  • the algae or fractions thereof used in the described invention may be heat treated.
  • the algae or a fraction thereof is sterilised before use in the culture medium of the invention.
  • Any suitable form of sterilisation may be used.
  • the sterilisation may be a heat treatment.
  • the sterilisation may comprise a chemical treatment and/or a radiation treatment
  • the algae or a fraction thereof may be sterilised before it is added to the medium.
  • the algae or a fraction thereof may be added to the culture medium and the combination of culture medium and algae (or a fraction thereof) may be sterilised before addition of the LAB and culturing.
  • Sterilisation may be carried out by heat treatment. In one embodiment the sterilisation may be carried out by heating the sample to 100°C or more. In a preferred embodiment sterilisation of the algae or a fraction thereof, or algae (or a fraction thereof) and culture medium, is carried out at 100-130°C.
  • Sterilisation preferably is carried out for at least 5 minutes.
  • the sterilisation may be carried out for 10-30 minutes.
  • sterilisation of the algae or a fraction thereof, or algae (or a fraction thereof) and culture medium is carried out at 100-130°C for 10-30 minutes.
  • sterilisation of the algae or a fraction thereof, or algae (or a fraction thereof) and culture medium occurs at 1 15-125 °C for 15-25 minutes.
  • sterilisation of the algae, or algae and culture medium occurs at 121 °C for 20 minutes.
  • the algae, in particular spirulina, chlorella or a fraction thereof, is used is present or is added to the culture medium in which the LAB composition is cultured under aerobic growth conditions.
  • said algae, in particular spirulina, chlorella or a fraction thereof is present or is added to the culture medium, at a concentration of at least 0.1 g/L, at least 0.2 g/L, at least 0.3 g/L, at least 0.4 g/L, at least at least 0.5 g/L, in particular at least 1 g/L.
  • Concentrations of algae are given herein as gram (g) of algae or a fraction thereof by litre (L) of culture medium, before inoculation of a culture medium with LAB.
  • said algae (or a fraction thereof), in particular spirulina, chlorella or a fraction thereof is present or is added to the culture medium at a concentration of at least 2 g/L.
  • said algae (or a fraction thereof), in particular spirulina, chlorella or a fraction thereof is present or is added to the culture medium at a concentration of at least 5 g/L.
  • Said algae (or a fraction thereof), in particular spirulina, chlorella or a fraction thereof, can be present or be added to the culture medium at a concentration as high as 20, 30, 40, 50, 60, 70, 80, 90 or 100 g/L.
  • said algae (or a fraction thereof), in particular spirulina, chlorella or a fraction thereof is present or is added to the culture medium from a concentration selected from the group consisting of 0.1 , 0.5, 1 and 2 g/L to a concentration selected from the group consisting of 10, 20, 30, 40, 50, 60, 70, 80, 90 and 100.
  • said algae in particular spirulina, chlorella or a fraction thereof, is present or is added to the culture medium at a concentration from 0.1 to 10 g/L.
  • said algae in particular spirulina, chlorella or a fraction thereof, is present or is added to the culture medium at a concentration from 0.5 to 10 g/L.
  • said algae (or a fraction thereof), in particular spirulina, chlorella or a fraction thereof is present or is added to the culture medium at a concentration from 1 to 10 g/L. In a particular embodiment, said algae (or a fraction thereof), in particular spirulina, chlorella or a fraction thereof, is present or is added to the culture medium at a concentration from 1 to 5 g/L.
  • said algae (or a fraction thereof), in particular spirulina, chlorella or a fraction thereof is present or is added to the culture medium at a concentration from 2 to 10 g/L. In a particular embodiment, said algae (or a fraction thereof), in particular spirulina, chlorella or a fraction thereof, is present or is added to the culture medium at a concentration from 5 to 10 g/L. In a particular embodiment, said algae (or a fraction thereof), in particular spirulina, chlorella or a fraction thereof, is present or is added to the culture medium before inoculation of the LAB into said culture medium.
  • said algae in particular spirulina, chlorella or a fraction thereof, is added to the culture medium after inoculation of the LAB into said culture medium, but before culturing said LAB composition.
  • Lactic Acid Bacteria In a particular embodiment of any use or method of the invention, the term "lactic acid bacteria” (LAB) refers to any bacteria which produce lactic acid as the end product of carbohydrate fermentation. In a particular embodiment, the LAB is selected from the group consisting of species Streptococcus, Lactococcus, Lactobacillus, Leuconostoc, Pediococcus and Bifidobacterium or any combination thereof.
  • LAB composition it is meant a composition comprising at least one LAB strain, or a defined composition comprising two or more LAB strains or an undefined composition of LAB strains, which has been treated in accordance with the present invention to increase viable active biomass.
  • harvested LAB composition it is meant a LAB composition as defined herein which has further undergone harvesting methods as described herein.
  • said harvested LAB composition is obtained after separating the LAB from the final culture medium, such as at least 80% of the final culture medium is removed.
  • said harvested LAB composition is obtained after separating the LAB from a portion of the algae or a fraction thereof such that at least 5% of the algae or a fraction thereof initially admixed in step (i) is removed.
  • the LAB composition or harvested LAB composition comprises a pure LAB strain, or a defined composition of pure LAB strains or an undefined composition of pure LAB strains.
  • the LAB composition or harvested LAB composition consists of a strain from species Streptococcus, Lactococcus, Lactobacillus, Leuconostoc, Pediococcus or Bifidobacterium, or comprises at least a strain from species Streptococcus, Lactococcus, Lactobacillus, Leuconostoc, Pediococcus or Bifidobacterium.
  • the LAB composition or harvested LAB composition consists of a strain from species Lactococcus or a strain from species Leuconostoc, or comprises at least a strain from species Lactococcus or at least a strain from species Leuconostoc.
  • the LAB composition or harvested LAB composition consists of a strain from species Streptococcus thermophilus or comprises at least a strain from Streptococcus thermophilus.
  • at least one strain/' or "at least a strain” it is meant 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 strains, or even more.
  • the LAB composition or harvested LAB composition comprises a strain of species Lactococcus, in particular a strain of Lactococcus lactis, in particular a strain of Lactococcus lactis ssp. lactis or a strain of Lactocococcus lactis ssp cremoris.
  • the LAB composition or harvested LAB composition is a mixture of more than one LAB for example of a) at least one strain of species Lactococcus, in particular a strain of Lactococcus lactis, in particular a strain of Lactococcus lactis ssp.
  • lactis or a strain of Lactocococcus lactis ssp cremoris, and b) at least one strain selected from the group consisting of Lactobacillus, Leuconostoc, Pediococcus, Bifidobacterium and any combination thereof.
  • the LAB composition or harvested LAB composition consists of several strains of the species Lactococcus, in particular of several strains of Lactococcus lactis, in particular of several strains of Lactococcus lactis ssp. lactis or of several strains of Lactocococcus lactis ssp cremoris.
  • severeal strains it is meant at least 2, in particular 2, 3, 4, 5, 6, 7, 8, 9 or 10 strains, or even more.
  • the LAB composition or harvested LAB composition consists of at least one strain, in particular one strain or several strains, of Lactococcus lactis ssp.
  • the LAB composition or harvested LAB composition consists of a strain of species Lactococcus, in particular a strain of Lactococcus lactis, in particular a strain of Lactococcus lactis ssp. lactis or a strain of Lactocococcus lactis ssp cremoris.
  • the LAB composition or harvested LAB composition comprises or consists of the Lactococcus lactis strain DGCC2631 (deposited at DSMZ under accession number DSM27777) and/or the Lactococcus lactis strain DGCC9209 (deposited at DSMZ, under accession NO.DSM12015).
  • the LAB composition or harvested LAB composition comprises or consists of a strain of species Leuconostoc, in particular Leuconostoc mesenteroides.
  • the LAB composition or harvested LAB composition comprises or consists of several strains of the species Leuconostoc.
  • the LAB composition or harvested LAB composition is a mix of a) at least one strain of species Leuconostoc, in particular a strain of Leuconostoc mesenteroides, and b) at least one strain selected from the group consisting of Lactobacillus, Pediococcus, Bifidobacterium and any combination thereof.
  • the LAB composition comprises or consists of the Leuconostoc mesenteroides strain DGCC 4634.
  • Any LAB composition or harvested LAB composition as described herein within the invention may also comprise additional microorganism(s), prokaryotic or eukaryotic, such as yeast, mould or other bacteria, for example Corynebacterium species, Staphylococcus species.
  • a LAB composition or harvested LAB composition of the invention may also comprise remaining algae or a fraction thereof, and/or the culture media.
  • a LAB composition or harvested LAB composition comprises or consists of several LAB strains, or LAB strain(s) and additional microorganism(s)
  • the parameters defined herein are determined for the whole LAB composition after culture, or for the whole harvested LAB composition.
  • a sample of a LAB composition or a harvested LAB composition is any fraction of the LAB composition or harvested LAB composition. For example, 0.1 %. 0.2%, 0.5% or 1 % weight by weight of the composition. It may be dried and or powdered, and/or liquid and/or concentrated.
  • a "LAB culture medium” is a LAB admixed with a culture medium to form a LAB composition which is defined as a composition not treated with algae (or a fraction thereof) in accordance with the present invention.
  • a “harvested LAB culture medium” is a LAB admixed with a culture medium to form a LAB composition which is defined as a composition not treated with algae (or a fraction thereof) in accordance with the present invention, which has been cultured under aerobic growth conditions and has further undergone harvesting methods as described herein.
  • a LAB culture medium may comprise any LAB, and may for example comprise any of the LAB strains of the LAB composition.
  • a LAB culture medium is a control, it is cultured under the same conditions as the LAB composition of the invention (in particular regarding aerobic conditions), and comprises initially the same LAB strains, but is cultured in the absence of algae.
  • sample sized sample herein is meant a sample obtained in an identical way of the same weight and/or volume.
  • a LAB composition of the invention and a LAB culture medium are compared by any test, a same-sized sample is used.
  • the concentration of the LAB in the culture medium before culture or the concentration of LAB added to the culture medium before culture is in the range of 10 5 to 10 10 CFU (colony forming units) per ml of the culture medium.
  • Optical density can be used to evaluate the biomass of a culture.
  • Optical density (OD) increase or "gain" between the beginning of a culture and the end of a culture is positively correlated to the cell biomass production.
  • a high OD gain corresponds to a high bacterial cell concentration in the composition.
  • OD gain of a LAB composition cultured in the presence of said algae under aerobic conditions it is meant the difference between the optical density (OD) of a LAB composition after culture in the presence of said algae (or a fraction thereof) under aerobic conditions and the OD of the same LAB composition before culture in the presence of said algae [also called lactic acid bacteria admixture].
  • OD gain of the LAB culture medium cultured in the absence of said algae it is meant the difference between the OD of the LAB culture medium after culture in the absence of said algae (or a fraction thereof) under aerobic conditions minus the OD of the same LAB culture medium before culture in the absence of said algae (or a fraction thereof).
  • ratio delta OD algae / delta OD control (expressed in %) is the ratio of the OD gain as defined above for a LAB composition after culture under aerobic conditions in presence of the algae or a fraction thereof, over the OD gain as defined above for a lactic acid bacteria culture medium cultured under the same aerobic conditions in absence of algae or a fraction thereof (control).
  • the increased viable active biomass of the LAB composition of the invention may further be defined as an optical density (OD) gain of the LAB composition of at least 120% the OD gain of the LAB culture medium, or a same sized sample thereof, obtained after culture in the absence of said algae (or a fraction thereof).
  • said OD gain of a LAB composition of the invention is at least 130, at least 140, at least 150, at least 200 or at least 300 % the OD gain of the LAB culture medium (cultured in the absence of said algae or a fraction thereof).
  • the OD is determined at a wavelength of 400-800nm, suitably 500-700nm, most suitably 600-700nm.
  • the OD is determined at a wavelength of 660nmm.
  • OD may be determined using a spectrophotometer, for example using a Genesys 20TM Spectrophotometer (Thermo Scientific).
  • Spectrophotometer Thermo Scientific
  • An example of assay to determine the delta OD of a culture is given in Assay III below.
  • Assay I is a milk activity test, which may be used to determine the Ta of any cultured LAB. In this case it is used to determine the Ta of the LAB composition of the invention compared to the Ta of the LAB culture medium (control).
  • Milk acidifying activity of the biomass of the LAB composition obtained after culture under aerobic conditions [or the LAB culture medium - control] is qualified on CINAC equipment using a multi pH probe recorder. Thus, pH decrease is recorded using CINAC equipment from Alliance Instruments which is 32 channel pH probe recorder.
  • the milk substrate used is prepared by dissolving skim milk powder (for example skim milk from Humana) into deionised water at a 9/100 dilution (for example 90gr of skim milk powder into 91 Ogr of water). Rehydration takes place for 30 minutes at room temperature under stirring conditions, and is followed by sterilising the reconstituted milk in 480ml volume at 96 °C for 30min.
  • the milk substrate is pre warmed at 30°C and then inoculated at 0.1 % weight by weight with a non pH control culture of a LAB composition [obtained after culture of LAB under aerobic growth conditions either in absence of algae or a fraction thereof (control), in presence of algae or a fraction thereof, or in presence of hemin].
  • pH is recorded for 16h at 30°C.
  • Ta in minutes is recorded, and corresponds to the time in minutes for the LAB composition after culture [or the LAB culture medium - control] to decrease the milk pH by 0.08 units. This time period is called lag phase.
  • Assay II is used to calculate the CFU (number of colony forming units) of any cultured LAB. In this case it is used to determine the CFU of the LAB composition of the invention compared to the CFU of the LAB culture medium (control).
  • 1 ml of LAB composition of the invention (obtained after culture under aerobic growth conditions in presence of algae or a fraction thereof), or 1 ml of LAB culture medium (obtained after culture under aerobic growth conditions in absence of algae or a fraction thereof - control), or in 1 ml of LAB culture medium cultured in the additional presence of hemin, is diluted by mixing in 9ml of TS tryptone salt diluent at room temperature to obtain a dilution 10-1 after mixing on a vortex for 30s; this dilution procedure is repeated by inoculation of 1 ml of dilution 10-1 into 9 ml of TS diluent till 10-8.
  • Proper inoculums of dilutions 10-7 and 10-8 are then plated on M17 agar using Eddy jet spiral inoculator's and incubated under anaerobic conditions at 30°C for 48h. At the end of incubation, grown colonies are enumerated and CFU is determined after multiplication by the dilution factor.
  • Assay III is used to calculate the optical density (OD) of any cultured LAB.
  • 1 ml of LAB composition of the invention (obtained after culture under aerobic growth conditions in presence of algae or a fraction thereof), or 1 ml of LAB culture medium (obtained after culture under aerobic growth conditions in absence of algae or a fraction thereof- control), or in 1 ml of LAB culture medium cultured in the additional presence of hemin, is diluted into 9, 18 or 27 ml of distilled water in order to obtain a diluted sample, and an OD range comprised between 0.1 and 0.5 on diluted sample.
  • the diluted sample is then measured at 660nm, using a Genesys 20TM spectrophotometer (with a spectral slit width specification of 8nm and an effective optical path of 10mm); the OD (Optical Density) is calculated by multiplying the dilution factor of the bacterial culture by the value given by the spectrophotometer. Calculated OD is the multiplication of the dilution factor by the value given by the spectrophotometer.
  • a LAB composition as defined herein which has further undergone harvesting methods as defined herein is a "harvested LAB composition”.
  • Harvesting involves separating the LAB of the LAB composition from at least a percentage of the final culture medium, and/or from at least a portion the algae (or a fraction thereof).
  • the LAB may be isolated from the final culture medium and/or from algae (or a fraction thereof).
  • the culture is stopped and harvesting occurs when the LAB concentration in the final culture medium (or LAB composition) reaches a sufficiently high concentration, in particular reaches a concentration of 10 8 to 10 12 CFU / ml of the LAB composition, most suitably at least 10 9 , 10 10 , 10 11 or 10 12 CFU/ml of the LAB composition.
  • harvesting is carried out using any conventional techniques enabling the separation of the LAB in the LAB composition from the final culture medium and/or the algae or a fraction thereof.
  • harvesting concentrates the LAB (and thus a harvested LAB concentrate (e.g. a harvested LAB composition concentrate) is obtained).
  • Harvesting and concentration may use, but is not limited to, any appropriate technology of sedimentation or flocculation, in particular by centrifugation and/or membrane filtration and/or ultrafiltration. Alternatively, concentration may optionally be carried out as a separate, additional step.
  • the harvesting step implements any conventional methods, including the ones cited above, in order to concentrate the harvested LAB.
  • the LAB in the harvested LAB composition may reach a concentration of 10 8 to 10 13 CFU, or 10 8 to 10 12 CFU/g / of the harvested LAB composition.
  • the LAB in the harvested LAB composition may reach a concentration of at least 10 8 , at least 10 9 , at least 10 10 , at least 10 11 or at least 10 12 CFU/g of the harvested LAB composition.
  • the LAB are concentrated in the harvested LAB composition by a factor of 5-40 fold.
  • the concentration factor is defined as the ratio between the resulting harvested LAB composition and the non-harvested LAB composition from which it is harvested.
  • the LAB are concentrated by a factor of 10-30 fold.
  • the LAB are concentrated by a factor of 15- 25 fold.
  • final culture medium it is meant the water and water-soluble components of the culture medium at the end of the culture (i.e., when one decides to stop the growth of the LAB).
  • Water-soluble components include for example residual substrates and metabolites obtained following the culture of the LAB (such as for example lactate).
  • Harvesting produces a harvested LAB composition with lower amounts of final culture medium and/or algae, and a higher concentration of LAB.
  • the harvesting step comprises or consists of separating the LAB from the final culture medium, such as at least 80% of the final culture medium is removed, to produce the harvested LAB composition. In a particular embodiment, at least 85% of the final culture medium is removed. In a particular embodiment, at least 90% of the final culture medium is removed. In a particular embodiment, at least 95% of the final culture medium is removed. This produces a harvested LAB composition with a higher concentration of LAB compared with the LAB composition.
  • the final culture medium may be separated from the LAB composition to produce the harvested LAB composition, using any conventional techniques including in particular by centrifugation and/or filtration, and/or membrane filtration and/or ultrafiltration.
  • Harvesting and concentrating may also involve purifying. Purifying may involve removing the colour, particularly the green colour, of the algae (or a fraction thereof), from the harvested LAB. This separation in one embodiment excludes drying.
  • the harvesting step comprises or consists of separating the LAB from the final culture medium, such as at least 5% of the algae or a fraction thereof present during culturing is removed, to produce the harvested LAB composition.
  • at least 10% of the algae or a fraction thereof is removed.
  • at least 20% of the algae or a fraction thereof is removed.
  • at least 30% of the algae or a fraction thereof is removed.
  • at least 40% of the algae or a fraction thereof is removed.
  • at least 50% of the algae or a fraction thereof is removed.
  • at least 60% of the algae or a fraction thereof is removed.
  • Preferably at least 70% of the algae or a fraction thereof is removed.
  • at least 80% of the algae or a fraction thereof is removed.
  • at least 90% of the algae or a fraction thereof is removed.
  • Preferably 100% of the algae or a fraction thereof is removed.
  • 5-100% of the algae or a fraction thereof is removed. In one embodiment 5-90% of the algae or a fraction thereof is removed. In one embodiment 5- 80% of the algae or a fraction thereof is removed. In one embodiment 5-70% of the algae or a fraction thereof is removed. In one embodiment 5-60% of the algae or a fraction thereof is removed. In one embodiment 5-50% of the algae or a fraction thereof is removed. In one embodiment 5-40% of the algae or a fraction thereof is removed. In one embodiment 5-30% of the algae or a fraction thereof is removed. In one embodiment 5-20% of the algae or a fraction thereof is removed. In one embodiment 5- 10% of the algae or a fraction thereof is removed. In one embodiment 10-100% of the algae or a fraction thereof is removed.
  • 10-90% of the algae or a fraction thereof is removed. In one embodiment 10-80% of the algae or a fraction thereof is removed. In one embodiment 10-70% of the algae or a fraction thereof is removed. In one embodiment 10-60% of the algae or a fraction thereof is removed. In one embodiment 10-50% of the algae or a fraction thereof is removed. In one embodiment 10-40% of the algae or a fraction thereof is removed. In one embodiment 10-30% of the algae or a fraction thereof is removed. In one embodiment 10-20% of the algae or a fraction thereof is removed.
  • the harvested LAB composition may comprise algae (or a fraction thereof), media and extracts of said algae (or a fraction thereof) and media. In one embodiment, the harvested LAB composition does not comprise all of the algae or a fraction thereof present in the final culture medium and/or LAB composition.
  • the harvested LAB composition is preferably a concentrated harvested LAB composition.
  • Algae (or a fraction thereof) may be removed from the LAB composition to produce the harvested LAB composition, using any conventional techniques including in particular by centrifugation and/or filtration, and/or membrane filtration and/or ultrafiltration.
  • Harvesting and concentrating may also involve purifying. Purifying may involve removing the colour, particularly the green colour, of the algae (or a fraction thereof), from the harvested LAB. The removal of the algae or fraction thereof may improve the quality of the product obtained following the addition into a fermentable substrate of said harvested LAB composition (or intermediate starter culture or starter culture as defined herein obtained from said harvested LAB composition).
  • the removal of the algae or fraction thereof may improve the quality of food products or feed products, in particular of the fermented products, in particular fermented food products or fermented feed products, in particular in particular dairy products, obtained using said harvested LAB composition (or intermediate starter culture or starter culture as defined herein obtained from said harvested LAB composition).
  • the LAB in particular the harvested LAB concentrate, is maintained as a liquid or in a liquid form.
  • the invention disclosed herein also includes LAB intermediate starter cultures, methods of producing said intermediate starter cultures and uses of said intermediate starter cultures.
  • intermediate starter culture of the invention as used herein it is meant one harvested LAB composition obtained following the implementation of the culturing and harvesting methods described herein, which has further undergone washing and/or freezing and/or drying and/or formulating.
  • intermediate starter culture as used herein it is meant one harvested LAB composition obtained by a culturing method, followed by a harvesting step, which has further undergone washing and/or freezing and/or drying and/or formulating.
  • Intermediate starter culture and “intermediate starter culture of the invention” do not have an identical meaning to "starter culture” as defined below.
  • One or more intermediate starter cultures may be mixed to form a starter culture, as discussed below.
  • said intermediate starter culture of the invention has a concentration of LAB which is between 10 7 to 10 12 or 10 8 to 10 13 CFU/g, and more preferably at least at least 10 7 , at least 10 8 , at least 10 9 , at least 10 10 , at least O or at least 10 12 CFU/g of the intermediate starter culture.
  • the LAB intermediate starter culture of the invention may be produced, obtained or obtainable from any harvested LAB or harvested LAB composition of the invention.
  • the intermediate starter culture of the invention is obtained from the harvested LAB or harvested LAB composition, after washing the LAB from a harvested LAB composition of the invention, and/or freezing and/or drying a harvested LAB composition of the invention and/or formulating a harvested LAB composition of the invention.
  • washing refers to the use of water or other liquid to remove any components from the LAB composition or harvested LAB composition of the invention.
  • the harvested LAB composition is washed to form an intermediate starter culture.
  • the intermediate starter culture of the invention is obtained from the harvested LAB or harvested LAB composition as described above, by freezing and/or drying a harvested LAB or harvested LAB composition of the invention.
  • the LAB intermediate starter culture of the invention may be, liquid, solid, frozen, dried, freeze dried, in the form of pellets or frozen pellets, or in a powder or dried powder.
  • the intermediate starter culture of the invention is frozen, dried, freeze-dried, in the form of pellets or frozen pellets, or in a powder or dried powder.
  • the LAB, the harvested LAB, the harvested LAB composition or LAB concentrate as obtained in any embodiments defined above, in particular the LAB concentrate, is frozen, dried (e.g. spray dried) or freeze-dried in particular to obtain the intermediate starter culture of the invention as defined herein, preferably to obtain intermediate starter culture of the invention as frozen LAB pellets or as a powder or dried LAB powder.
  • cryoprotectant(s) and/or stabilizer(s) is/are mixed with the harvested LAB before freezing.
  • Cryoprotectants or stabilizers include, but are not limited to, glucose, lactose, raffinose, sucrose, trehalose, adonitol, starch maltodextrin, glycerol, mannitol sorbitol, plolypropylene glycol, polyethylene glycol, ribitol alginate, bovine serum albumin, carnitine, citrate, cystein, dextran, dimethyl sufoxide, sodium glutamate, glycin, betaine, glycogen, hypotaurin, skimmed milk peptone, polyvinyl pirrolidine, taurine, nucleosides, nucleotides and any combination thereof.
  • the LAB in the frozen, dried, or freeze dried intermediate starter cultures may be in a concentration of at least 10 8 , at least 10 9 , at least 10 10 , at least 10 11 or at least 10 12 CFU/g of the intermediate starter culture.
  • the intermediate starter culture of the present invention comprises between about 0.1 % and 20% of remaining algae or a fraction thereof, calculated as weight by weight of the intermediate starter culture, when this intermediate starter culture is frozen.
  • the invention is also directed to a frozen intermediate starter culture as defined herein, comprising between about 0.1 % and 20% of remaining algae or a fraction thereof, calculated as weight by weight of the intermediate frozen starter culture.
  • the frozen intermediate starter culture of the present invention comprises between about 0.5% and 15% of remaining algae or a fraction thereof, calculated as weight by weight of the frozen intermediate starter culture.
  • the frozen intermediate starter culture of the present invention comprises between about 0.5% and 10% of remaining algae or a fraction thereof, calculated as weight by weight of the frozen intermediate starter culture
  • remaining algae or a fraction thereof
  • remaining chlorella may mean remaining spirulina or remaining chlorella.
  • the term "remaining algae” as used herein means the algae or a fraction thereof that was present during the culturing step and which remains in the LAB composition or the harvested LAB composition or the intermediate starter culture or the frozen intermediate starter culture.
  • the invention disclosed herein also includes a dried intermediate starter culture obtained following drying of the frozen intermediate starter culture as defined herein, in particular following drying of the frozen intermediate starter culture of the present invention comprises between about 0.1 % and 20% of remaining algae or a fraction thereof, calculated as weight by weight of the intermediate starter culture.
  • said LAB intermediate starter culture is in the form of frozen pellets or a dried powder.
  • said LAB intermediate starter culture preferably obtained or obtainable by the method above, has a concentration of LAB of at least 10 7 CFU/g of the intermediate starter culture, in particular of at least 10 8 , at least 10 9 , at least 10 10 , at least 10 11 , or at least 10 12 CFU/g of the intermediate starter culture, and comprises remaining algae (or a fraction thereof), in particular remaining spirulina, remaining chlorella or a fraction thereof, and is optionally of green colour.
  • said LAB intermediate starter culture comprises remaining spirulina or a fraction thereof and has a green colour.
  • the intermediate starter culture in particular in the form of pellets, has a colour which is not white to beige colour.
  • the intermediate starter culture, in particular in the form of pellets has a similar colour as the pigments of the algae or the fraction thereof.
  • the intermediate starter culture, in particular in the form of pellets has a green colour.
  • the intermediate starter culture, in particular in the form of pellets has a red colour.
  • the intermediate starter culture in particular in the form of pellets, has a white to beige colour.
  • Any method such as washing or enzymatic treatment to remove partially or totally the pigments contained in the algae (or a fraction thereof) or the extract may be used in order to obtain a intermediate starter culture with a white to beige colour.
  • These methods of pigment removal may be implemented at any stage of the method to produce a intermediate starter culture, in particular before adding the algae or the fraction thereof with the LAB composition, or after harvesting the LAB composition to obtain a harvested LAB composition or an intermediate starter culture.
  • the invention also includes LAB starter cultures, methods of producing said starter cultures and uses of said starter culture.
  • starter culture as used herein it is meant a mixture comprising or consisting of at least one intermediate starter culture of the invention as defined herein with at least one other intermediate starter culture (The other intermediate starter culture may be any intermediate starter culture, which is an intermediate starter culture of the invention or not). Each intermediate starter culture is produced by culturing separately, wherein at least one of the intermediate starter cultures is produced by the methods of the invention described herein.
  • the starter culture comprises or consists of a mixture of one intermediate starter culture of the invention and one other intermediate starter culture. In a particular embodiment, the starter culture comprises or consists of a mixture of one intermediate starter culture of the invention and two other intermediate starter cultures.
  • the starter culture is a mixture comprising or consisting of two intermediate starter cultures of the invention. These two intermediate starter cultures of the invention are different from each other, in that they are cultured separately from each other using the methods of the invention.
  • the starter culture of the invention comprises or consists of a mixture of one intermediate starter culture of the invention and two or more other intermediate starter cultures.
  • the starter culture is a mixture comprising two or more different intermediate starter cultures of the invention.
  • the intermediate starter cultures mixed to form the starter culture of the invention comprise different species, and/or different strains, and/or different combinations of species and strains of LAB.
  • the intermediate starter cultures mixed to form the starter culture of the invention comprise at least one different species, and/or at least one different strain, and/or at least one different combination of species and strains of LAB.
  • the intermediate starter cultures mixed to form the starter culture of the invention each comprise the same species, and/or strains, and/or combinations of species and strains of LAB.
  • the intermediate starter cultures mixed to form the starter culture of the invention each comprise at least one of the same species, and/or least one of the same strains, and/or least one of the same combinations of species and strains of LAB.
  • the starter culture of the invention may be frozen, dried, freeze dried, liquid, solid, in the form of pellets or frozen pellets, or in a powder or dried powder.
  • the starter culture of the invention is frozen, dried, freeze-dried, in the form of pellets or frozen pellets, or in a powder or dried powder.
  • a starter culture according to the invention is obtained by mixing several (at least 2, preferably 2 or 3) intermediate starter cultures, at least one being an intermediate starter culture of the invention.
  • the invention also includes a method to manufacture a starter culture comprising mixing at least one intermediate starter culture of the invention and at least one intermediate starter culture.
  • the at least one intermediate starter culture of the invention and at least one intermediate starter culture are mixed under frozen form, in the form of pellets or frozen pellets.
  • the at least one intermediate starter culture of the invention and at least one intermediate starter culture are mixed under dried form, freeze-dried form, in the form of a a powder or dried powder.
  • intermediate starter culture or starter culture provides the use of an intermediate starter culture or starter culture as defined above or as obtained or obtainable by any of the method as defined herein,
  • the intermediate starter culture or starter culture of the invention may preferably be used for increasing the acidity of a fermentable substrate and/or for producing a fermented product. Adding a intermediate starter culture or starter culture to a fermentable substrate (such as a fermentable medium) may also be referred to as seeding a substrate or medium, or seeding a substrate or medium to be fermented.
  • the invention also concerns the use of an intermediate starter culture or starter culture of the invention for preparing products, in particular food products or feed products, in particular fermented products, in particular fermented food products or fermented feed products.
  • the invention also provides a method for preparing a product, preferably a food or feed product, in particular fermented products, in particular fermented food products or fermented feed products, comprising a) seeding a medium with an intermediate starter culture or starter culture of the invention, b) optionally fermenting said medium, and c) obtaining said product.
  • a product preferably a food or feed product, in particular fermented products, in particular fermented food products or fermented feed products, comprising a) seeding a medium with an intermediate starter culture or starter culture of the invention, b) optionally fermenting said medium, and c) obtaining said product.
  • said medium is milk, in particular milk of animal origin.
  • said medium is milk, in particular milk of animal origin
  • the fermented product is a dairy product, preferably a yoghurt, a cheese (such as an acid curd cheese, a hard cheese, a semi-hard cheese, a cottage cheese), a butter, a buttermilk, quark, a sour cream, kefir, a fermented whey-based beverage, a koumiss, a milk beverage, a yoghurt drink, a fermented milk, a matured cream, a fromage frais, a milk, a dairy product retentate, a processed cheese, a cottage cheese, a cream dessert, or infant milk.
  • the invention also concerns said product, in particular dairy products obtained by said preparation method.
  • the term "product” as used herein may also include such as meat products (such as salami or sausages), animal feed and pet food; surface coating material (such as paint), agricultural products (such as crops and seeds), feed bakery, wine, beer or other beverages, or vegetable products and pharmaceutical industry products.
  • the intermediate starter cultures or starter cultures of the invention may also be used to produce proteins including enzymes and various kinds of useful compounds.
  • a "food” or “feed” as used herein may be an animal or human food or feed.
  • Formulating The LAB composition, harvested LAB composition, intermediate starter cultures and/or starter culture of the present invention may be formulated into any suitable form.
  • Formulating may include pelleting, capsules, caplets, tableting, blending, coating, layering, formation into chewable or dissolvable tablets, formulating into dosage controlled packets, formulating into stick packs and powdering.
  • Formulating may also include the addition of other ingredients to the LAB composition, harvested LAB composition, intermediate starter cultures and/or starter culture of the present invention.
  • Suitable ingredients include for example food ingredients, sugars, carbohydrates, and dairy products.
  • formulating does not include the addition of any further LAB.
  • the LAB composition, harvested LAB composition, intermediate starter culture and/or starter culture of the present invention may be packaged.
  • packaging occurs after harvesting the LAB composition. In one embodiment, packaging occurs after freezing the harvested LAB composition. In one embodiment, packaging occurs after drying the harvested LAB composition. In one embodiment, packaging occurs after mixing intermediate starter culture(s).
  • the packaging may be comprised of a vacuum pack, sachet, box, a blister pack, stick pack, or tin.
  • Probiotics The LAB composition or harvested LAB composition of the invention may be used as, or as components of, probiotics and/or prebiotics.
  • a non-digestible food ingredient that beneficially affects the host by selectively stimulating the growth and/or the activity of one or a limited number of beneficial bacteria.
  • probiotic as used herein defines live microorganisms (including bacteria or yeasts for example) which, when for example ingested or locally applied in sufficient numbers, beneficially affects the host organism, i.e. by conferring one or more demonstrable health benefits on the host organism. Probiotics may improve the microbial balance in one or more mucosal surfaces.
  • the mucosal surface may be the intestine, the urinary tract, the respiratory tract or the skin.
  • probiotic as used herein also encompasses live microorganisms that can stimulate the beneficial branches of the immune system and at the same time decrease the inflammatory reactions in a mucosal surface, for example the gut.
  • At least 10 6 -10 12 Whilst there are no lower or upper limits for probiotic intake, it has been suggested that at least 10 6 -10 12 , preferably at least 10 6 -10 10 , preferably 10 8 -10 9 CFU/ml as a daily dose will be effective to achieve the beneficial health effects in a subject.
  • the LAB composition and the harvested LAB composition of the invention may be used a probiotics or prebiotics.
  • the LAB composition or harvested LAB composition may be added to other components to create composition for use as probiotics and/or prebiotics. They may be used a direct fed microbials in food or feed for humans or animals (such as cattle).
  • the probiotics and/or prebiotics may be used as a food or feed additive for humans or animals (such as cattle).
  • the invention also provides a method for preparing a probiotic or prebiotic product, preferably a food or feed product for humans or animals. This method comprises creating a LAB composition of the invention, or harvested LAB composition of the invention, and using this as a probiotic or prebiotic, either directly or in a composition.
  • the probiotics and prebiotics of the invention may be frozen, dried, freeze dried, liquid, solid, in the form of pellets or frozen pellets, or a in powder or dried powder.
  • the invention may also be described by the following numbered paragraphs:-
  • a method of producing a lactic acid bacteria composition with increased lactic acid bacteria viable active biomass comprising the steps of
  • said increased viable active biomass is defined as at least one of the following: a) the lag phase of growth on a fermentable substrate (Ta value) of the lactic acid bacteria composition, or a sample thereof, is at least 10 minutes less than the Ta value of a lactic acid bacteria culture medium, or a same sized sample thereof, obtained after culture in the absence of said algae; and/or b) the content of viable cells, expressed in colony forming units (CFU), of the lactic acid bacteria composition, or a sample thereof, is at least 120% the content of viable cells of a lactic acid bacteria culture medium, or a same sized sample thereof, obtained after culture in the absence of said algae.
  • Ta value lag phase of growth on a fermentable substrate
  • CFU colony forming units
  • a value of the lag phase of growth on a fermentable substrate (Ta value) of the lactic acid bacteria composition, or a sample thereof is at least 10 minutes less than the Ta value of a lactic acid bacteria culture medium, or a same sized sample thereof, obtained after culture in the absence of said algae; and/or b) wherein the content of viable cells, expressed in colony forming units (CFU), of the lactic acid bacteria composition, or a sample thereof, is at least 120% the content of viable cells of a lactic acid bacteria culture medium, or a same sized sample thereof, obtained after culture in the absence of said algae.
  • CFU colony forming units
  • said algae is a prokaryotic algae, such as a cyanobacteria, or an eukaryotic algae selected from the group consisting of a red algae and a green algae, or any fraction thereof, and more particularly wherein said algae is selected from the group consisting of spirulina, chlorella and a fraction thereof.
  • the Ta value of a lactic acid bacteria composition obtained after culture in the presence of said algae under aerobic conditions is at least 12, at least 15, at least 20, at least 30 or at least 40 minutes less than the Ta value of a lactic acid bacteria culture medium obtained after culture in the absence of said algae.
  • optical density (OD) gain of the lactic acid bacteria composition is of at least 120, at least 130, at least 140, at least 150, at least 200 or at least 300 % the OD gain of a lactic acid bacteria culture medium or a same sized sample thereof obtained after culture in the absence of said algae.
  • the OD gain is determined by Assay III as presented herein. 8. The method or use according to any preceding numbered paragraph wherein said algae is used or is present or is added into said culture medium, at a concentration of at least 0.1 g/L, at least 0.5 g/L, in particular at least 1 g/L of culture medium.
  • said lactic acid bacteria composition consists of a strain from species Lactococcus or a strain from species Leuconostoc, or comprises at least a strain from species Lactococcus or at least a strain from species Leuconostoc.
  • a lactic acid bacteria starter culture obtained by the method or use of any one of numbered paragraphs 1 -1 1 .
  • a lactic acid bacteria starter culture according to numbered paragraph 12 which comprises at least 0.1 % of remaining algae or a fraction thereof, calculated as weight by weight of the starter culture.
  • a lactic acid bacteria starter culture according to any of numbered paragraphs 12 to 13 , which is frozen, dried, freeze dried, liquid, solid, in the form of pellets or frozen pellets, or a powder or dried powder.
  • a method of producing a harvested lactic acid bacteria composition comprising the steps of
  • said increased viable active biomass of the LAB composition is defined as at least one of the following: a) the lag phase of growth on a fermentable substrate (Ta value) of the lactic acid bacteria composition, or a sample thereof, is at least 10 minutes less than the Ta value of a lactic acid bacteria culture medium, or a same sized sample thereof, obtained after culture in the absence of said algae or a fraction thereof; and/or b) the content of viable cells, expressed in colony forming units (CFU), of the lactic acid bacteria composition, or a sample thereof, is at least 120% the content of viable cells of a lactic acid bacteria culture medium, or a same sized sample thereof, obtained after culture in the absence of said algae or a fraction thereof;
  • Ta value the lag phase of growth on a fermentable substrate
  • CFU colony forming units
  • step iii) harvesting the LAB from the LAB composition of step ii) by separating the LAB from the final culture medium, such as at least 80% of the final culture medium is removed to produce a harvested LAB composition.
  • a method of producing a harvested lactic acid bacteria composition comprising the steps of
  • a method of producing a harvested lactic acid bacteria composition comprising the steps of (i) admixing a culture medium and a lactic acid bacteria, and an algae or a fraction thereof;
  • said increased viable active biomass of the LAB composition is defined as at least one of the following: a) the lag phase of growth on a fermentable substrate (Ta value) of the lactic acid bacteria composition, or a sample thereof, is at least 10 minutes less than the Ta value of a lactic acid bacteria culture medium, or a same sized sample thereof, obtained after culture in the absence of said algae or a fraction thereof; and/or b) the content of viable cells, expressed in colony forming units (CFU), of the lactic acid bacteria composition, or a sample thereof, is at least 120% the content of viable cells of a lactic acid bacteria culture medium, or a same sized sample thereof, obtained after culture in the absence of said algae or a fraction thereof;
  • Ta value the lag phase of growth on a fermentable substrate
  • CFU colony forming units
  • said increased viable active biomass of the LAB composition is defined as at least one of the following: a) the lag phase of growth on a fermentable substrate (Ta value) of the lactic acid bacteria composition, or a sample thereof, is at least 10 minutes less than the Ta value of a lactic acid bacteria culture medium, or a same sized sample thereof, obtained after culture in the absence of said algae or a fraction thereof; and/or b) the content of viable cells, expressed in colony forming units (CFU), of the lactic acid bacteria composition, or a sample thereof, is at least 120% the content of viable cells of a lactic acid bacteria culture medium, or a same sized sample thereof, obtained after culture in the absence of said algae or a fraction thereof;
  • Ta value the lag phase of growth on a fermentable substrate
  • CFU colony forming units
  • step (iii) harvesting the LAB from the LAB composition of step ii) by separating the LAB from a portion of the algae or a fraction thereof such that at least 5% of the algae or a fraction thereof initially admixed in step (i) is removed, to produce a harvested LAB composition.
  • a method of producing a harvested lactic acid bacteria composition having increased lactic acid bacteria viable active biomass comprising the steps of
  • said increased viable active biomass of the LAB composition is defined as at least one of the following: a) the lag phase of growth on a fermentable substrate (Ta value) of the lactic acid bacteria composition, or a sample thereof, is at least 10 minutes less than the Ta value of a lactic acid bacteria culture medium, or a same sized sample thereof, obtained after culture in the absence of said algae or a fraction thereof; and/or b) the content of viable cells, expressed in colony forming units (CFU), of the lactic acid bacteria composition, or a sample thereof, is at least 120% the content of viable cells of a lactic acid bacteria culture medium, or a same sized sample thereof, obtained after culture in the absence of said algae or a fraction thereof;
  • Ta value the lag phase of growth on a fermentable substrate
  • CFU colony forming units
  • step iii) harvesting the LAB from the LAB composition of step ii) by separating the LAB from the final culture medium, such as at least 80% of the final culture medium is removed to produce a harvested LAB composition.
  • Lactococcus lactis DGCC2631 Three lactic acid bacteria strains have been studied: the Lactococcus lactis DGCC2631 , the Lactococcus lactis DGCC9209 and the Leuconostoc mesenteroides DGCC4634.
  • Lactococcus lactis DGCC2631 has been deposited under the Budapest Treaty on September 24, 2013 in the name of Danisco Deutschland GmbH at the Leibniz-lnstitut DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen, GmbH (Inhoffenstr. 7B, D-38124 Braunschweig), under number DSM 27777.
  • the depositor (Danisco GmbH) authorises the applicant to refer to the deposited biological material in the application, and gives unreserved and irrevocable consent to the deposited material being made available to the public.
  • the following algae have been assayed, at a concentration of 1 , 2 and 5g/L of growth medium:
  • Chlorelle Chlorella Setalg A416 081 1 N vulgaris
  • a stock solution of hemin at 0.05 % is prepared by dissolving 100 mg of hemin (Sigma, product number 51280, porcine origin) in 2 ml of 5N NaOH, to which 198 ml of water is then added; the solution is autoclaved at 120°C for 20 minutes.
  • Control is the media used in the LAB culture medium (control). 1 .4. Culture conditions
  • Each cultured LAB is tested to qualify its ability to acidify milk.
  • the milk substrate used is prepared by dissolving 90gr of skim milk powder from Humana into deionised water at a 9/1 00 dilution (for example 90gr skim milk powder into 91 Ogr of water. Rehydration takes place for 30mn at room temperature under stirring conditions and is followed by sterilising the reconstituted milk in 480ml volume at 9Q°C for 30min. Milk activity tests are pre warmed at 30°C and then inoculated at 0.1 % weight by weight with the LAB composition of the invention. pH is recorded for each milk test, for 16h at 30°C, using CINAC equipment from Alliance Instruments which is 32 channel pH probe recorder.
  • the time (minutes) to decrease the pH by 0.08 pH units is calculated for each sample. Comparison is made to a LAB composition which is identical except it is cultured without algae (the LAB culture medium (control)), or hemin culture (control plus hemin, as described in 1 .3 above). In this experiment the LAB is not treated in any way after culture before it is used to measure the Ta. However, if the culturing is carried out in a bioreactor, or any liquid form, separation of the LAB, such as by centrifugation, may be necessary before the Ta measurement may be carried out.
  • 1 ml of LAB composition is diluted by mixing in 9ml of TS tryptone salt diluent at room temperature to obtain a dilution 1 0-1 after mixing on a vortex for 30s; this dilution procedure is repeated by inoculation of 1 ml of dilution 1 0-1 into 9 ml of TS diluent till 10- 8.
  • Proper inoculums of dilutions 1 0-7 and 10-8 are then plated on M1 7 agar using Eddy jet spiral inoculator's and incubated under anaerobic conditions at 30°C for 48h. At the end of incubation, grown colonies are enumerated and CFU (colony forming unit) final result determined after multiplication by the dilution factor.
  • the CFU of the LAB culture medium (control cultured under the same aerobic conditions) and hemin culture are also measured in the same way. 1 .7. OD measurement
  • 1 ml of LAB composition is diluted into 9ml, or in 18 or in 27 ml of distilled water in order to obtain a diluted sample (and an OD range comprised between 0.1 and 0.5 on diluted sample).
  • the diluted sample is then measured at 660nm in a Thermo Scientific Genesys 20TM spectrophotometer (with a spectral slit width specification of 8nm and effective optical path of 10mm); the OD (Optical Density) is calculated by multiplying the dilution factor of the bacterial culture by the value given by the spectrophotometer. Calculated OD is the multiplication of the dilution factor by the value given by the spectrophotometer.
  • the OD of the LAB culture medium (control cultured under the same aerobic conditions) and hemin culture are also measured in the same way.
  • Results are provided in Figures 1 to 6, and Tables 1 to 1 1 .
  • Tables 1 to 3 and 5 to 1 1 describe, from the left to the right: the nature of the cultured LAB, the nature of the added algae [hemin means that hemin is added instead of algae; control means that no compound (no algae, no hemin) is added, i.e. this is the LAB culture medium], the supplier (if needed), the concentration (if needed), and the 6 following parameters: delta OD (or OD gain), which corresponds to the difference between the OD of the cultured LAB at the end of the culture under aerobic conditions minus the OD of the same contents (LAB in media plus algae or hemin or neither as listed in the table) before culture;
  • Ta which corresponds to the time (in minutes) necessary for the cultured LAB obtained after culture under aerobic conditions, to decrease the pH of the milk by 0.08 pH units, as per the above;
  • ratio delta OD with algae over delta OD control (in %), which corresponds to the ratio of (1 ) the delta OD of a LAB composition of the invention (cultured under aerobic conditions in the presence of the algae) over (2) the delta OD of a control (the LAB culture medium as defined above - LAB cultured in the same conditions, same LAB, same growth conditions).
  • the ratio corresponds to the ratio of the delta OD of a culture grown under aerobic conditions in the presence of hemin over the delta OD of a control culture grown in the same conditions.
  • the control delta OD used for these calculations is the one described in the same Table.
  • a percentage above 120% means that addition of the algae has a significant effect on the OD increase;
  • the difference corresponds to the difference between the Ta of a LAB composition after culture under aerobic conditions in the presence of hemin minus the Ta of a control LAB after culture in the same conditions.
  • the control Ta used for these calculations is the one described in the same Table.
  • a difference more than 10 minutes means that addition of the algae has a significant effect on Ta reduction. This means at least 5%, at least 10%, at least 15%, at least 20% difference compared to the control.
  • ratio CFU with algae over CFU control (in %), which corresponds to the ratio of the CFU of a the LAB composition of the invention over the CFU of the LAB culture medium (control) composition
  • the ratio corresponds to the ratio of the CFU of a LAB composition after culture under aerobic conditions in the presence of hemin over the CFU of a control culture grown in the same conditions.
  • the control CFU used for these calculations is the one described in the same Table. A percentage above 120% means that the addition of the algae has a significant effect on the CFU increase (20% more than the control).
  • the Ta and CFU values for the same cultures but under anaerobic culture conditions are also given in Tables 1 -1 1 .
  • spirulina at 1 to 5gr/L increases the OD gain between 21 1 and 262% as compared to a control culture, confirming that spirulina increases the biomass of Lactococcus lactis.
  • addition of spirulina at 1 to 5gr/L increases the CFU, between 155 and 280% as compared to a control culture, confirming that the increased biomass of Lactococcus lactis following spirulina addition is a viable biomass.
  • the Ta in milk of a LAB composition of the invention cultured with spirulina, is reduced between 28 and 56 minutes, confirming that the addition of spirulina not only increases the biomass of viable Lactococcus lactis, but also increases the biomass of viable active Lactococcus lactis.
  • control culture is the LAB culture medium as previous defined.
  • Spirulina is a blue-green algae (cyanobacteria). Further assays have been done on the same Lactococcus lactis strain, with 3 different algae: one green algae (Chlorella) and 2 brown algae (Laminaria and Fucus). SEAH2 is the same spirulina as the one used in Table 1 .
  • Lactococcus lactis Lactococcus lactis.
  • the Ta of a LAB composition of the invention cultured with chlorella addition is reduced between 20 and 28 minutes, confirming that the addition of chlorella not only increases the biomass of viable Lactococcus lactis, but also increases the biomass of viable active Lactococcus lactis.
  • Table 4 clearly shows that, in absence of LAB, the ratio of OD gain is reduced as compared to a control (a LAB culture medium as previous defined).
  • the Ta is increased from at least 300 minutes (up to 648 minutes) as compared to a control.
  • the same type of results has been obtained with both spirulina and chlorella.
  • the Ta of a LAB composition of the invention cultured with spirulina addition is reduced between 24 and 60 minutes, as compared to a control.
  • control culture is the LAB culture medium as previous defined.
  • control culture is the LAB culture medium as previous defined.
  • Tables 7 and 8 show that the addition of spirulina gives better results in terms of Ta reduction (36 versus 52 minutes) as compared to the addition of hemin, with the first Lactococcus lactis strain DBCC2631 .
  • Table 9 shows that for the other Lactococcus lactis, the addition of spirulina gives similar results as hemin addition (60-minute Ta reduction).
  • Table 10 shows that the addition of chlorella gives similar results as hemin addition (-28 minutes versus -32 minutes for Ta measurement).
  • the addition of spirulina gives better results in terms of Ta reduction as compared to the addition of hemin (hemin increases the Ta as compared to the control).
  • spirulina a cyanobacteria
  • chlorella a green algae
  • cultures under anaerobic growth conditions were also carried out.
  • the same growth conditions apply (lactic acid bacteria strains, media type, inoculation rate, temperature, culture time etc), except that tests were run under static conditions [absence of oxygen].
  • Ta aerobic which corresponds to the time (in minutes) necessary for the cultured LAB obtained after culture under aerobic conditions, to decrease the pH of the milk by 0.08 pH units, as per the above ( e.g. as per Assay I);
  • CFU aerobic which corresponds to the colony-forming units obtained for the cultured LAB obtained after the culture under aerobic conditions, as described above (e.g. as per Assay II);
  • Ta anaerobic which corresponds to the time (in minutes) necessary for the cultured LAB obtained after culture under anaerobic conditions, to decrease the pH of the milk by 0.08 pH units, as per the above (e.g. as per Assay I);
  • CFU anaerobic which corresponds to the colony-forming units obtained for the cultured LAB obtained after the culture under anaerobic conditions, as described above (e.g. as per Assay II);
  • Tables 12 to 16 illustrate the Ta difference between LAB cultured under aerobic and anaerobic growth conditions.
  • the Ta difference is only the result of aeration [oxygen addition], and is comprised between -16 and +20 min.
  • aerobic conditions have no impact on Ta (difference is 0) (Table 15).
  • aerobic conditions increase the Ta by 20 minutes (Table 16).
  • the aerobic conditions decrease the Ta, even in absence of algae, but only between 4 and 16 minutes (Tables 12 to 14).
  • the use of algae in addition to aerobic growth conditions drastically decreases the Ta, as compared to culture with algae but in anaerobic conditions.
  • the decrease is up to -84 minutes (Table 13) as compared to anaerobic conditions, and is on average between 20 and 45 minutes (Tables 12 to 14).
  • the Ta decrease is comprised between 24 and 52 minutes, knowing that the aerobic conditions have a negligible or little impact on Ta in the control (Table 14).
  • DGCC4634 whereas aerobic conditions increase the Ta, both in the control and in presence of hemin, the addition of algae decreases the Ta between 8 and 12 minutes (Table 16).
  • delta Ta [delta Ta aerobic minus Ta anaerobic] were more negative (i.e., the Ta was shorter) in presence of algae than in absence of algae, what confirms the beneficial effect of algae under aerobic conditions on Ta reduction.
  • delta Ta [delta Ta aerobic minus Ta anaerobic] were identical or more negative (i.e., the Ta was identical or shorter) using algae, confirming the effect of using algae instead of hemin.
  • CFU increase, between LAB cultured with algae under aerobic growth conditions and LAB cultured with algae under anaerobic algae
  • Tables 12 to 16 illustrate the ratio of the CFU of LAB cultured under aerobic growth conditions over the CFU of LAB cultured under anaerobic growth conditions.
  • a ratio CFU aerobic over CFU anaerobic above 100% meaning more viable LAB after culturing in aerobic conditions than after culturing in anaerobic conditions
  • Aeration has a larger effect on DGCC9209 with an increase of the ratio CFU aerobic over CFU anaerobic of about 300% in the control (Table 15).
  • the use of algae in addition to aerobic growth conditions increases the ratio CFU aerobic over CFU anaerobic of at least 200% and up to 500% for DGCC2631 . This increase is in the same range as hemin (tables 12 to 14).
  • the increase of the ratio CFU aerobic over CFU anaerobic is similar to or a slightly higher than the control, and is between 235 and 325% for DGCC9209 (Table 15) and is about 140% for DGCC4634 (Table 16).

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Abstract

L'invention concerne un procédé pour produire une composition de bactéries d'acide lactique (LAB) ou une composition de LAB récoltée, ayant une biomasse active viable LAB accrue, à l'aide d'algues ou d'une fraction de ces dernières. Les algues peuvent être des algues procaryotes, telles que des cyanobactéries, ou des algues eucaryotes choisies parmi le groupe constitué d'algues rouges et d'algues vertes, ou d'une fraction de ces dernières, et sont en particulier choisies parmi le groupe constitué de spiruline, de chlorelle et d'une fraction de ces dernières.
PCT/EP2014/073505 2013-11-01 2014-10-31 Utilisation d'algues pour accroître la biomasse active viable de bactéries d'acide lactique WO2015063282A1 (fr)

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