EA039406B1 - Pasteurisation process for microbial cells and microbial oil - Google Patents

Abstract

An improved pasteurisation process for pasteurising microbial cells is disclosed. The process has three stages, a first heating stage, a second plateau stage at which the cells are held at a (maximum and) constant temperature, and a third cooling stage. Both the heating and the cooling stages are rapid, with the temperature of the cells passing through 40 to 80°C in no more than 30 min in the heating stage. The heating rate is at least 0.5°C/min and during cooling is at least -0.5°C/min. The plateau maximum temperature is from 70 to 85°C. By plotting the pasteurisation protocol on a time (t, min) versus temperature (T, °C) graph, one obtains a trapezium having an area less than 13,000°Cmin. Not only does this result in a smaller energy input (and so a reduction in costs), but a better quality (and less oxidised) oil results having a peroxide value (POV) of less than 1.5 and an anisidine value (AnV) of less than 1.0.

Description

The invention relates to a method for pasteurization of microbial cells, which includes heating the cells at a temperature in the range from 40 to 70°C for no more than 30 minutes. The heating rate during the pasteurization process may be at least 0.5° C./min. The pasteurization process may include three steps, namely a heating step, a plateau step (where the cells are kept at a constant temperature), and a cooling step. If you represent the pasteurization protocol graphically, then the area under the curve of time (in minutes) versus temperature (°C) is less than 13000°Cmin. After pasteurization, a polyunsaturated fatty acid (PUFA) such as arachidonic acid or oil can be extracted from the microbial cells. The oil may have a low peroxide value (POV) and/or a low anisidine value (AnV).

Introduction.

Polyunsaturated fatty acids, or PUFAs, are abundant in nature, and a wide variety of PUFAs are produced by various single-celled microorganisms (algae, fungi, etc.). One particularly important PUFA is arachidonic acid (ARA), which is one of a number of long chain polyunsaturated acids (LC-PUFAs). Chemically, arachidonic acid is cis-5,8,11,14-eicosatetraenoic acid (20:4) and belongs to the (n-6) LC-PUFA family.

Arachidonic acid is one of the main precursors of a wide variety of biologically active substances, collectively known as eicosanoids, a group including prostaglandins, thromboxanes and leukotrienes. Also, arachidonic acid is one of the components of the lipid fraction of human milk, and it is believed that it is necessary for the optimal development of the nervous system of infants. Arachidonic acid has a wide range of different uses, including use in infant formula, food, and pet food.

Application WO-A-97/37032 (Gist-Brocades) relates to the production of a PUFA-containing oil from pasteurized biomass of microbial cells. However, rapid heating to, or cooling from, the temperature at which pasteurization is carried out is not disclosed.

In addition, no importance is attached to the total amount of energy used during the pasteurization process.

Both applications WO-A-00/15045 and WO-A-01/67886 relate to the use of Mucorales fungi in food production. The first of these papers refers to the need to reduce the RNA content before incorporating cells into foods, and suggests the use of a heating step. One pasteurization or heat shock can be done. The second document proposes to eliminate the heating step in order to reduce the RNA content and replace it with keeping the fungal cells in the fermenter and maturation.

International Patent Application No. PCT/EP01/08902 relates to a process for preparing oil blends by combining crude ω ₆ and crude ω ₃ containing PUFA oil to form an oil blend and then purifying the crude oil blend.

Known methods include heating the biomass or microbial cells. It is also known from application no. WO-A-97/37032 that microbial cells can be pasteurized before the PUFA is extracted from them as an oil. However, the inventors of the present invention have found that the quality of the oil that is extracted from the pasteurized cells can be improved with the new pasteurization method. In particular, the resulting oil is less oxidizing or oxidizing and has low peroxide value (POV) and/or anisidine value (AnV). In addition, applicants have found that this new pasteurization process is more efficient because it requires less energy to carry out. Therefore, the method is advantageous because it can not only improve the quality of the oil, but also reduce its cost, since less energy is required.

Brief description of the figures

Fig. 1 is a graph of temperature (°C) versus time (min) for three pasteurization protocols (A and C according to the invention, B is shown for comparison);

fig. 2 is a plot of temperature (°C) versus time (min) for pasteurization at three different temperature plateaus (40, 70 and 85°C);

fig. 2 and 4 are plots of AnV value (and POV for FIG. 3) versus time (h);

fig. 5 is a plot of POV (meq/kg) and AnV versus temperature (°C) for pasteurization at two times (hold/plateau) (8 and 300 s);

fig. 6 and 7 are plots of temperature (°C) versus time (min) at two times (hold/plateau) (8 s for FIG. 6, 5 min for FIG. 7) at five different temperatures (60, 80, 100, 120 and 140°C).

Description of the invention

Therefore, the invention provides an improved process for pasteurizing microbial cells. In addition to less energy consumption, the pasteurization process according to the invention makes it possible to obtain a product of better quality.

- 1 039406

Thus, the first aspect of the present invention relates to a method for pasteurizing microbial cells, where the method includes heating the cells at a temperature (including) in the range from 40 to (60°C or) 70°C for no more than 30 minutes or heating the cells at a rate of at least 0.5°C/min. Therefore, this aspect provides rapid heating of microbial cells during pasteurization, and such a high heating rate has not previously been disclosed in this field. While pasteurization temperatures are reported in the art, the rate of heating is not evaluated or discussed, or the fact that this parameter is important, and a relatively high pasteurization rate may provide benefits. In fact, high heating rates are counter-intuitive since they can be expected to oxidize or otherwise destroy the PUVA or the oil that can be extracted from the cells.

The second aspect of the present invention relates to a method for pasteurization of microbial cells, where the method includes a pasteurization protocol comprising (at least) three stages. These are: a (first) heating step, a (second) plateau step (in which the microbial cells are kept at a desired temperature or where the cells are kept at a constant and/or maximum temperature), and a (third) cooling step. This aspect of the invention relates to a three-stage pasteurization protocol. If this protocol is presented as a graph of the dependence of time on temperature, then a trapezoid will be obtained.

A third aspect of the invention relates to a method for pasteurizing microbial cells, wherein the method includes applying a pasteurization protocol such that the area under the time (min) versus temperature (°C) curve is less than 13,000°C-min. In a graph of time versus temperature, area represents the amount of energy expended in heating the cells during the pasteurization process. It has been found that rapid heating and/or rapid cooling (which correspond to the first and third stages of the second aspect, respectively) can provide benefits such as better oil quality. In addition, the amount of energy required to carry out the pasteurization process can be reduced compared to the pasteurization methods described in the art. Therefore, this third aspect relates to the energy required to carry out the pasteurization process.

The fourth aspect of the invention relates to a method for pasteurizing microbial cells, where the method includes (heating the cells and further) keeping the cells at an elevated temperature (T, ° C) for a time (t, min), for example, at a plateau stage, where the value of the product tT (t i.e. the multiplication of time and temperature parameters, for example during the plateau stage) is in the range from 140 to 100800°C-min. As will be appreciated, this fourth aspect is similar to the second aspect in that it includes a plateau stage. In this case, the cells can be maintained at a constant or maximum temperature. Therefore, the product tT can be the area under the time-temperature curve for a given plateau stage.

The first aspect is rapid heating.

In this aspect, the cells are heated such that the temperature of the cells changes from 40 to 70° C. (or 60° C.) in no more than 30 minutes (eg, no more than 15 minutes). Preferably, the time required for heating from 40 to 70°C is no more than 40-50 minutes. Alternatively or additionally, the cells are heated at a rate of at least 0.5°C/min. Of course, microbial cells can start (or begin to heat up) at temperatures below 40°C. For example, the cells may be at room or ambient temperature. The cells may be at a fermentation temperature such as 30±5°C. Thus, the cells may be at a temperature in the range of 20 to 40°C such as 23-27°C (or 25-29°C to 32-37°C) when heating (pasteurization) starts. In some cases, microbial cells can be cooled, for example, after the end of fermentation. Thus, the cells may have (at start) a temperature in the range of 5 to 10° C., eg 7 to 9° C. when heating starts.

Microbial cells can be heated so that their temperature is above (60°C or) 70°C. Thus, this value may not be the final microbial cell temperature during pasteurization. In fact, cells can be heated to temperatures above (60°C or) 70°C. The temperature may rise to a value in the range of 70 to 90°C, 110 or 130°C, for example 75 to 87°C and optimally 78 to 84°C. Therefore, the maximum temperature during pasteurization may be within these limits, but for some embodiments may be as high as 100, 120 or 140°C. Preferably, the cells are maintained or maintained at a given (maximum) temperature.

Therefore, it is obviously clear that the cells can be heated at a temperature below or ranging from 40°C to a temperature of 70°C or higher. Limits from 40 to 70°C can provide a shot in a wider range of heating temperatures, for which it is possible (and therefore calculated) to determine the period of time (and, therefore, the speed).

It can be calculated that heating (from 40 to 70°C for 30 min) corresponds to a rate of 1°C/min. However, the rate may be somewhat lower than required, and in the first aspect, rapid heating means that the heating rate is above 0.5°C/min. Preferably the rate is at least 0.6, 1.0 or even 1.5°C/min. However, particularly high heating rates are envisaged, depending on the equipment and the volume or mass of microbial cells that are subjected to heating. Thus, heating rates above 2.0 or even 2.5° C./min are also within the scope of the invention.

Particularly high heating rates can be obtained using special equipment. It allows a high temperature to be reached in a short period of time and thus to minimize any oxidation or degradation of the PUFAs, or oils from the microbial cells, which can then be isolated. Thus, heating up to a maximum temperature of 140, 150 or even 160°C may take place. Preferably the heating is carried out to a temperature of from 100 to 180°C, for example from 120 to 160°C, preferably from 130 to 150°C. By using fast heaters, these temperatures can be achieved particularly quickly, for example in less than one minute (30 s). Similar temperatures can be reached within 20, 30, 40 and 50 seconds, or maintained within 150, 175, 200, 225 or 250 seconds. However, these temperatures can be reached so very quickly, for example within 2, 4, 6, 8 or 10 seconds, for example when using an infusion heater or when using relatively small samples. In this way, heating rates of up to 50, 100, 150 and even 200°C per minute can be achieved. Slightly lower heating rates are also possible, from 5-10°C to 50-60°C per minute, for example from 15 to 45°C per minute.

It has been found that this rapid heating during rapid pasteurization is not only more efficient and requires less energy, but has been found to be at least one factor responsible for producing better quality microbial cell oil (when extracted from cells after pasteurization). ).

The second aspect is the three-stage pasteurization protocol.

The first step may be a heating step. It actually corresponds to the rapid heating described in the first aspect of the invention, and therefore all the features and characteristics of the first aspect apply to the first stage (heating) of the second aspect, mutatis mutandis.

The second stage is the period when the cells are on a plateau (at temperature). Thus, the cells can be maintained at a certain desired temperature (within a tolerance of plus or minus 1, 2, 5 or even 10°C) for the desired period of time. Thus, the cells are maintained at a constant temperature. Preferably, this plateau temperature (or temperature limits) is the maximum temperature reached during pasteurization. The temperature at the plateau stage (and/or the maximum temperature during pasteurization) is preferably at least 70°C. It can be below 90 or 100°C, preferably in the range from 70 to 85°C, for example from 70 to 77°C. Alternatively, it may be in the range of 80-160°C, for example 100-140°C.

The duration of the plateau stage, or the period of time in which the cells are kept at the desired or maximum temperature, may be 5 seconds to 90 minutes, such as 1 or 10 to 80 minutes, such as 20 to 70 minutes. Optimally, this time period is from 40 or 50 to 60 or 70 minutes, for example from 45 to 65 minutes, preferably from 55 to 63 minutes. Short periods of time from 8 s to 5 min are also possible.

The third stage is the cooling stage. Preferably, the cells are cooled to a temperature that is similar to or within the range specified for the start of heating (or first step). Preferably, the microbial cells are cooled and/or heated linearly (in the first and/or third stages, respectively), i.e. when plotting time versus temperature, the cooling or heating mode is a (roughly) straight line. The cells are allowed to cool, or they can be actively cooled, for example by using a heat exchanger or coolants, for example (lowering) to or below ambient temperature or room temperature.

Preferably the cooling rate is at least 0.4, 0.6, 1.0 or 1.5°C/muh. These values represent the achievable cooling rates when the cells themselves are allowed to cool. However, higher cooling rates are possible, especially if active cooling is used. Thus, cooling rates of at least 2.0, 2.5, 3.0 and even 3.5°C/muh are achievable. However, higher cooling rates are possible, such as above 5°C/muh, for example from 7 or 10°C/muh to 50 or 60°C/muh, preferably from 15 to 45°C/muh.

The preferred heating and/or cooling rate is preferably maintained at least 10, 20 or 30°C/muh, although in some embodiments it can be achieved in the range of at least 40 to 50°C/muh.

Obviously, it is understood that with a fast heating step and a fast cooling step, the amount of energy used in pasteurization is reduced. This circumstance not only leads to cost savings, but also may not have a negative effect on the quality of the (final) oil from microbial cells, in fact it has a positive effect on the oil.

The third aspect is the area under the time-temperature curve (energy input).

From the second aspect, it becomes apparent that if the pasteurization protocol according to the invention is presented as a graph of time versus temperature, then it has the shape of a trapezoid. The first (heating) and third (cooling) stages may be triangular in shape, while the middle or second (plateau) stage (the subject of the fourth aspect) is (generally) a rectangle. The area under the time versus temperature curve represents the amount of energy put into the system. By dividing the pasteurization protocol into three parts, it is possible to calculate the area of the graph and, therefore, the energy costs.

In a third aspect, the area under the time (in minutes) versus temperature (in °C) curve is below 13,000°C-min. However, lower values are achieved and values lower than 11000, 10000, 9000, 8000 and even 1000°C-min are possible. In preferred aspects of the invention, these values may be no more than 7000, 6000 or 800°C-min. In the graph shown, time is plotted on the x-axis (or horizontal axis, or abscissa), and 0°C represents the starting point. Temperature will therefore be plotted on the y-axis (or vertical axis, or ordinate), and 0°C represents the reference point.

After heating the microbial cells to the pasteurization temperature, they can cool themselves (or they can be cooled). As a rule, the cells are cooled to room or ambient temperature, or at least to a temperature below 30°C. Therefore, there is a period of time not only for heating the cells to a temperature in the range of 30 to 60°C, but also a period of time for cooling the cells from 60 to 30°C. You can sum the two periods of time to obtain a total period of heating and cooling in the range of 30-60°C and back to 30°C. Preferably, this total time period is less than 150 minutes, such as less than 120 or 100 minutes. However, with smaller samples, shorter time periods can be achieved and the total time period (from 30 to 60°C and back to 30°C) can be less than 70, 50 and even 30 minutes.

The fourth aspect is the pasteurization protocol from the plateau stages.

This protocol can be one of the second aspect, where there is a (eg, first) heating stage and (eg, second) cooling stage and a middle plateau stage (eg, second or middle or intermediate). However, this is not mandatory and other pasteurization protocols are envisaged to be included. A fourth aspect relates to the preferred features of this plateau stage. All signs and characteristics of the second (and other) aspects refer to the fourth aspect with the appropriate, necessary changes.

All cells are supported and maintained at a certain desired temperature (with a tolerance of plus or minus 1, 2, 5 or even 10°C) for temperature (T,°C) for time (t, min). These two parameters can be multiplied to obtain the product tT. It advantageously ranges from 140 or 280 to 50,000 or 100,800° C.-min. Preferably, this product is from 500, 1000, 2000 or 3000 or even 6000 to 10000, 18000 or 25000°C-min. The optimal value of the product tT is in the range from 2000 to 6000, for example from 3000 to 5000, optimally from 4000 to 4500°C-min. In some embodiments, the product tT is from 13 to 900, for example from 100 or 200 to 700 or 800, optimally from 300 or 400 to 600 or 700° C.-min.

Thus, similarly to the third aspect, it is understood that the product tT is the area under the time-temperature curve of the cells when they are kept at elevated temperature. Thus, the product tT actually represents the area under the curve for the plateau stage only (not the heating or cooling stage).

PUFA extraction.

The fifth aspect of the present invention relates to a method for obtaining PUFA from microbial cells, where the method includes pasteurizing the cells according to one of the first, second, third or fourth aspects of the invention described earlier, and extracting and/or isolating PUFA from the pasteurized cells.

A sixth aspect of the present invention relates to a microbial cell oil which may contain at least 40% arachidonic acid (ARA) and/or may have a triglyceride content of at least 90%. The oil may have a POV value of less than 2.5, 1.5, 0.8, 0.6, or even 0.5, and/or an AnV value of less than 1.0. The oil is prepared by the method according to the fifth aspect.

Polyunsaturated fatty acids (PUFA) and oils from microbial cells.

The PUFA may be one PUFA or two or more different PUFAs. Each PUFA may be from the n-3 or n-6 family. Preferably it is C ₁₈ , C ₂₀ or C ₂₂ PUFA. It may be a PUFA with at least 18 carbon atoms and/or at least 3 or 4 double bonds. PUFA can be provided as a free fatty acid, a salt, as a fatty acid ester (eg, methyl or ethyl ester), as a phospholipid, and/or as a mono-, di, or triglyceride.

Suitable (n-3 and n-6) PUFAs include docosahexaenoic acid (DHA, 22:6 Ω 3) respectively from algae or fungi such as (dinoflagellates) Crypthecodinium or (fungi) Thraustochytrium;

γ-linolenic acid (GLA, 18:3 Ω 6);

α-linolenic acid (ALA, 18:3 Ω 3);

conjugated linolenic acid (octadecadienoic acid, CLA);

- 4 039406 dihomo-y-linolenic acid (DGLA, 20:3 Ω 6);

arachidonic acid (ARA, 20:4 Ω 6); and eicosapentaenoic acid (EPA, 20:5 Ω 3).

Preferred PUFAs include arachidonic acid (ARA), docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA) and/or γ-linolenic acid (GLA). In particular, ARA is preferred.

PUFAs can be produced by cells pasteurized by the method of the invention, such as microbial cells. The latter may be bacteria, algae, fungi or yeasts. Fungi are preferred, preferably of the order Mucorales, eg Mortierella, Phycomyces, Blakeslea, Aspergillus, Thraustochytrium, Pythium or Entomophthora. A preferred source of ARA is Mortierella alpina, Blakeslea trispora, Aspergillus terreus or Pythium insidiosum. The algae may be dinoflagellates and/or include Porphyridium, Nitszchia or Crypthecodinium (eg Crypthecodinium cohnii). Yeasts include those belonging to the genera Pichia or Saccharomyces such as Pichia ciferii. The bacteria may be members of the genus Propionibacterium. Oil from microbial cells can be liquid (at room temperature).

Preferably, most of the PUFA is in the form of triglycerides. Thus, it is preferred that at least 50%, eg at least 60% or optimally at least 70% of the PUFA is in the form of triglycerides. However, the amount of triglycerides can be higher, for example it can be at least 85%, preferably at least 90%, optimally at least 95 or 98% oil. Of these triglycerides, it is preferred that at least 40%, eg at least 50% and optimally at least 60% of the PUFA is in the α-position of the glycerol (found in the backbone of the triglyceride), also known as the 1- or 3-position. Preferably, at least 20%, eg at least 30%, optimally at least 40% of the PUFA is in the β(2)-position.

The microbial cell oil may contain at least 10%, 35%, 40%, or 45% or more of the desired PUFA, such as arachidonic acid. It may have a triglyceride content of at least 90%. Preferably, the microbial oil has a triglyceride content in the range of 90 to 100%, such as at least 96%, preferably at least 98%, more preferably at least 99%, and optimally above 99.5%. Typically, the microbial cell oil will have an eicosapentaenoic acid (EPA) content below 5%, preferably below 1%, and more preferably below 0.5%. The oil may each have a _C20 , C20 _:3 , C22 _:0 and/or _C240 polyunsaturated fatty acid (PUFA) content of less than 5%, less than 2%, less than 1%. The content of free fatty acids (FFA) can be < 0.4, 0.2 or 0.1. The oil may include little or no GLA and/or DGLA.

The microbial cell oil may be a crude oil. It can be extracted from cells using a solvent such as supercritical carbon dioxide, hexane, or isopropanol.

pasteurization method.

As a rule, pasteurization is carried out after the end of fermentation. In a preferred embodiment, pasteurization will complete the fermentation because the heat during pasteurization will cause cell death. Therefore, pasteurization can be applied to fermentation broth (or cells in a liquid (aqueous) medium), although it can be subjected to microbial biomass obtained from the broth. In the latter case, pasteurization can take place while the microbial cells are still in the fermenter. Preferably, pasteurization takes place prior to any subsequent processing of the microbial cells, such as granulation milling (eg extrusion) or kneading.

Preferably, the pasteurization protocol is sufficient to inhibit or inactivate one or more enzymes that can adversely affect or degrade PUFA or microbial oil, such as lipase.

After fermentation is complete, the fermentation broth may be filtered or otherwise treated to remove water or aqueous liquid. After removing the water, a dense sediment of the biomass can be obtained. If pasteurization has not taken place, then the dehydrated cells (or solid biomass cake) can then be pasteurized.

Extraction method for PUFA.

The PUFA (or oil, typically containing PUFA) can then be extracted from the (pasteurized) microbial cells. Preferably it is extracted from (eg dried) granules (eg extrudates) containing cells. Extraction can be carried out using a solvent. Preferably a non-polar solvent is used, for example a C1-8, preferably a _{C2-6 alkane} , for example hexane. Carbon dioxide can be used (in liquid form, eg supercritical).

Preferably the solvent is allowed to percolate through the dried granules. Suitable methods for granulation and extrusion of microorganisms and subsequent extraction of oil from microbial cells,

- 5 039406 containing PUFA, described in the application WO-A-97/37032.

The solvent recovers the crude oil containing PUFA. This oil can be used in this state without further processing, or it can be subjected to one or more stages of refining. Typically, however, the crude oil contains a solvent, such as a solvent used to extract the oil (eg, hexane or an alcohol such as isopropyl alcohol), or an oil that has not undergone one (or preferably all) of the subsequent refining steps. Suitable refining methods are described in International Patent Application No. PCT/EP01/08902 (the contents of this document and all others provided herein are incorporated herein by reference). For example, the oil may be subjected to one or more refining steps, which may include acidification or degumming, alkaline treatment or removal of free fatty acids, bleaching or removal of pigments, filtration, fractionation by cooling (or cooling, for example, to remove saturated triglycerides), deodorization ( or removal of free fatty acids) and/or clarification by filtration (or removal of oil-insoluble substances). All these purification steps are described in more detail in PCT/EP01/08902 and can be applied to the steps described in this application with appropriate, necessary modifications.

The resulting oil is particularly suitable for food purposes and can be added to food (for humans) or feed (for animals). Examples include milk, infant formula, health drinks, bread, and pet food.

microbial cells.

The microbial (or microbial) cells used in the present invention may be any of those previously described, especially in the section on PUFAs and microbial cell oils. They may include, or be capable of producing, a PUFA or an oil, and accordingly the PUFA-containing oil can be extracted or isolated from the cells. They may be in filamentous form, like fungi or bacteria, or as single cells, like yeast, algae, and bacteria. The cells may include microorganisms such as yeast, fungi, bacteria, or algae. Preferred fungi are those belonging to the order Mucorales, for example, the fungus may be that of the genera Mortierella, Phycomyces, Blakeslea or Aspergillus. Preferred fungi are Mortierella alpina, Blakeslea trispora and Aspergillus terreus species.

With regard to yeasts, they are preferably those belonging to the genera Pichia (eg Pichia ciferrii species) or Saccharomyces.

The bacteria may be members of the genus Propionibacterium.

If the cells are derived from algae, they are preferably dinoflagellates and/or they belong to the genus Crypthecodinium. Algae of the species Crypthecodinium cohnii are preferred.

Heating.

It can be carried out by heating (cells) directly or indirectly. If the heating is direct, it can be carried out by passing steam into the fermenter. In an indirect way, the medium can be affected by a heat exchanger, fermenter walls or heating coils, or an external heat exchanger such as a plate heat exchanger.

Generally, pasteurization takes place in the fermenter in which the fermentation is carried out. However, for some microorganisms (such as bacteria) it is often preferable to first remove the cells from the tank and then pasteurize. Pasteurization may take place prior to other microbial treatments, such as drying or granulation.

As a result of pasteurization, as a rule, most or, if not all, microorganisms die. After pasteurization, at least 95, 96 or even 98% of all microorganisms die, i.e. which become unsustainable.

Acidification.

In some cases, it is desirable to reduce the risk of growth of pasteurized cells. One possibility is to acidify the cells with a suitable acid. Thus, to prevent the growth of microorganisms, it may be desirable to bring the cells to a pH value in the range of 3-4. However, wider pH ranges can be used depending on the cells and thus the pH can be adjusted to 2-5, optimally up to a range of about 3.3 to 3.7.

Acidification of cells can be carried out before pasteurization. However, it is preferable to carry it out afterwards.

The pH value can be adjusted by any means or by any suitable acid. It is preferred to carry it out using phosphoric acid, such as 85%, or dilute 55% or 33% phosphoric acid.

peroxide value (POV).

Preferably the POV of the microbial oil is in the range of 4 to 8 or 12, especially for crude oil. However, POV can be no more than 3.0, 2.5, or 2.0. However, significantly lower POV values can be obtained using the method of the invention, and these values can be below 1.5 or below 1.0. You can get values below 0.8 or 0.6 and even below 0.4. POV values (from embodiments) range from 1.3 (or 0.8) to 0.4. Peroxide number POV

- 6 039406 is usually expressed as meq/kg.

Anisidine number (AnV).

This number is an indicator of the content of aldehydes. Preferably, the anisidine value of the microbial oil is from 5, 6, 7, or 10 to 15, 20, or 25, especially for the crude oil. Advantageously, the AnV is at most 20, for example at most 15. It may be at most 10 or even at most 5. Preferably, the POV and/or AnV values refer to a crude oil rather than a refined one. AnV values (in the preferred examples) range from 15 to 5, optionally from 12 to 7.

Unrefined oils versus refined oils.

Some differences between these two oils are presented below. Each crude or refined oil may have one or more features in the following table for the crude or refined oil, respectively. Typically, crude oil contains an antioxidant (eg, tocopherol, ascorbyl palmitate)

Compound Preferred (for crude oil) unrefined butter Refined oil Unsaponifiables < 3.5% (w/w) 2.5% (w/w) 1.8 (w/w) Solvent (e.g. hexane) < 2000 million' ¹ 100-2000 million' ¹ Not detected or < 1 million' ¹ Phospholipids, % 2-3.5 0.05 Free < 1% 0.2% 0.08% fatty acids such as oleic acid pov <10 meq/kg 6 meq/kg 1.4 meq/kg Insoluble substances < 0.5% 0.1% - Phosphorus < 1000 mg/kg 5 mg/kg Silicon < 500 million' ¹ 100 million' ¹ 24 million ¹ Arsenic <0.5 mg/kg < 0.04 mg/kg <0.5 mg/kg Cadmium < 0.2 mg/kg <0.02mg/kg <0.1 mg/kg Mercury < 0.4 mg/kg < 0.4 mg/kg < 0.04 mg/kg Lead <0.1 mg/kg <0.1 mg/kg <0.1 mg/kg Copper < 0.2 mg/kg < 0.2 mg/kg < 0.02 mg/kg Moisture and volatiles < 1.0% 0.5 <0.02% Phosphatides (P/ppm ^-1 ) 50-100 < 10

Accordingly, the crude oil of the present invention may have one or more of the following:

(a) the content of unsaponifiable substances from 2.0 to 3.5% (wt./wt.);

(b) a solvent content (eg hexane) from 10, 50 or 100 ^ppm to 1000, 1500 or 2000 ^ppm ;

(c) a free fatty acid content of 0.1 or 0.2 to 1%, for example 0.2-0.6 or 0.3-0.5%;

(d) a POV value of 2, 3, 4, or 6 to 10;

(e) a phosphorus content of at least 2, 3 or 5 mg/kg;

(f) a silicon content of 50 or 100 to 500 ^ppm ; and/or (g) a water content of less than 1%, or from 0.5 to 1 or 2%.

Applications of oils and PUFA.

The sixth aspect of the invention relates to a composition containing the oil according to the fifth aspect, and where one or more other (additional) substances are suitable. The composition may be a food product and/or a food supplement for animals or humans. In embodiments of the invention that are intended for human consumption, the oils can be provided as suitable for human consumption, typically by refining or refining the oil derived from microbial cells.

The composition may be an infant formula or food product (for humans). In this case, the composition of the milk formula can be adjusted so that it will include similar amounts of lipids or PUFA that are found in regular human milk.

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This may include mixing the microbial cell oil of the invention with other oils to obtain an appropriate composition.

The composition may be a food or supplement for animals and fish. Such feeds and additives can be fed to any farm animal, in particular sheep, cattle and poultry. In addition, feeds or supplements can be fed to aquatic organisms intended for breeding, such as fish and armored aquatic animals. Thus, the composition may contain one or more feed substances or ingredients for such animals.

The oil according to the invention can be sold directly as an oil and in appropriate packaging, usually in disposable aluminum bottles coated on the inside with an epoxyphenol varnish and purged with nitrogen. The oil may contain one or more antioxidants (eg tocopherol, vitamin E, palmitate), each eg at a concentration of 50 to 800 ^ppm , eg 100 to 700 ^ppm .

Suitable compositions may include pharmaceutical or veterinary compositions, for example for oral administration, or cosmetic compositions. The oil may be taken as such or may be encapsulated, for example in a shell, and thus may be in the form of capsules. The shell or capsules may contain gelatin and/or glycerin. The composition may include other ingredients such as flavoring agents (eg, lemon and lime flavors and flavors), or a pharmaceutically or veterinarily acceptable carrier or excipient.

The preferred features and characteristics of one aspect of the invention apply to another aspect, mutatis mutandis.

The invention will now be described by way of examples, with reference to the following examples, which are provided for illustration and are not intended to limit the scope of the invention.

Example 1

I believe that the oxidation during production of oil from microbial cells containing PUFA, occurs as a result of enzymatic activity. Pasteurization has been considered as a method to reduce the chance of oxidation during the processing of microbial cells to produce oil from them. It was found that the degree of stabilization depends on the pasteurization conditions.

Therefore, a number of experiments were carried out to determine which pasteurization conditions can affect the level of oxidation and, in particular, the value of the peroxide value (POV) of the oil. The peroxide value was determined using the standard methodology detailed in AOCS:Cd8-53.

When setting up the experiments, the following protocol was followed: fermentation, storage, pasteurization, extraction (oils from microbial cells), oil analysis.

Fungi Mortierella alpina were cultivated in a fermenter. Fermentation was carried out for about 148 hours. M. alpina produced a PUFA called arachidonic acid (ARA). The biomass was removed from the fermenter and stored (below -18°C).

M. alpina biomass samples were taken from the fermentation broth while it was stored in the fermenter and immediately frozen.

Various pasteurization protocols were tested. Pasteurization was carried out at three different temperatures, namely 40, 70 and 85°C. Three stages followed, with a first rapid heating stage followed by a plateau stage (second or middle stage) at the desired temperature, which was the maximum temperature used. This was followed by a stage of rapid cooling (third). Various biomass samples were at the middle stage (plateau) for three different time periods, namely 1, 2 and 24 hours.

After pasteurization, an oil was obtained from microbial cells using wet extraction. This biomass sample was filtered, squeezed (under pressure) and the oil was extracted.

Then, the oil from the microbial cells was analyzed, mainly the peroxide value (POV) was determined using the AOCS method. In some samples, the content of ARA was determined. The results of the analysis showed that the oil obtained from microbial cells contained approximately 420 g of ARA per kg.

Detailed protocol: fermentation and extraction samples.

l fermentation broth was removed from the fermenter and filtered (Seitz filter, 2 l, FFA10). Then the resulting precipitate was washed with 600 ml of demineralized water. The crude cake was dried for 1 min and then pressed (using a HAFICO™ apparatus, COAO21 tincture press, 300-400 atm) at 400 bar. The crude extrudate was then used to extract oil from the microbial cells with 500 ml of hexane (Merck) at room temperature (20-25° C.) for 1 hour using an Ultra Turrax™ machine. Then the hexane was poured off. Then the remaining precipitate was washed with 250 ml of fresh hexane (with stirring for 30 min) at room temperature. Hexane was decanted and then added to the portion of hexane previously used for extraction.

The extract was then filtered using a glass filter in combination with a GFA glass filter. Hexane was then evaporated using a Rotavapor™ from the clear extract at about 50° C. for about 15 minutes. The oil was then transferred into sealed beakers, and each sample beaker was purged with nitrogen for 30 s. Then the beakers with samples were closed and stored at -18°C.

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pasteurization protocols.

Three different protocols were tested (A, B and C). Each consisted of three stages, a first heating stage, a second plateau stage (at maximum temperature), and a third cooling stage. In table.

protocols of three modes of pasteurization are presented.

Table 1

Time (t, min) Temperature (T, °C) for time (t) Stage Temperature change per stage (°C) Time per stage (min) Area under the curve (°C-min) Heating/cooling rate (°C/min) Time to pass 40°C/70°C (min) Combined period 40°C-70°C40°C (min) Area under the t versus T curve (°C-min) Mode A 0 75 135 210 25 70 70 25 Heating Pasteurization Cooling 45 0 45 t load = 75 t pasteur .= 60 t cool. = 75 1687.5 4200 1687.5 0.6 0 0.6 fifty fifty 100 7575 Mode B (outside the scope of the invention, for comparison) 0 102 25 72 Heating Pasteurization Cooling 48 t load = 102 t pasteur.= 60 t cool. = 198 4896 4320 4752 0.46 0 0.22 65.11 135 200.11 13968 Mode C 0 25 85 105 7 70 70 8 Heating Pasteurization Cooling 63 0 62 t load = 25 t pasteur.= 60 t cool. = 20 787.5 4200 620 2.52 0 3.10 11.90 9.68 21.58 5607.5

Additionally, the three pasteurization modes A, B and C are represented graphically in FIG. 1. Obviously, it is clear that it is possible to calculate the area under the curve of temperature (T, ° C) versus time (t, min) for each of the three stages for each mode and then summarize to obtain the total area under the curve for each of the three modes. These calculations are additionally given in Table. 1 above.

The peroxide value (POV) was determined for the oils obtained from cell extraction after three pasteurization protocols A, B, and C. The POV values of the extracted oils were 8.7, 14.3, and 2.4, respectively. Mode B had a low heating rate and a low cooling rate and was included for comparison purposes only. As a result, he gave the highest POV value of 14.3.

In contrast, both modes A and C were within the scope of the invention. Mode A had a higher heating rate and cooling rate in the first and third stages compared to mode B. Preferably, according to the invention, the heating and cooling rates are at least those presented for mode A. Mode A gave a POV value of 8.7.

However, the best results were obtained with mode C, in which the POV value was only 2.4. As can be seen from FIG. 1, in this case there was a very fast heating step and a fast cooling step (third).

Example 2

The experiments were carried out analogously to example 1, except that the pasteurization temperature values were in a wider range, namely, they were 40 ° C (for comparison), 70 and 85. The temperature regime (° C) depending on time (min) is presented in fig. 2 and in table. 2 below. The curve is basically the same for all four samples, but of course with increasing pasteurization plateau (from 1 to 4 or 24 hours) respectively.

table 2

40°C 70°C 85°С Time Temperature Time Temperature Time Temperature 0 100 0 8.0 0 7.0 ten 27.0 ten 55.0 ten 40.0 20 40.1 17 70.0 20 66.0 25 40.0 77 68.2 thirty 79.0 40 41.4 82 44.3 40 83.5 fifty 41.0 87 31.3 100 79.8 80 38, 7 92 21.8 105 55.3 85 27.5 97 16.0 110 38.7 90 19.3 102 9.7 115 26, 3 95 14.5 120 21.0 100 9.7 125 15.2 ON 7.5 130 11.3

Tested samples from two different fermentations (both with M. alpina) of different duration. Samples No. 11-20 in the table. 3 had a slightly longer fermentation, where about 2 m ^{3 of} broth was transferred to the inoculation fermenter and the fermentation was carried out for 48 hours without additional addition of glucose.

After pasteurization, the samples were processed starting with filtration under a nitrogen pressure of about 1 bar. Then the resulting precipitate was washed with industrial water (approximately 0.6 of the initial volume

-9039406 broth). Dehydration was carried out using a fruit press with a piston pressure of 300-400 bar. Then 500 ml fresh hexane was added and mixed for 1 minute using an Ultra Turrax for 1 minute. Then extraction was carried out for 1 hour at room temperature. After filtration, the resulting precipitate was washed with 250 ml of fresh portion of hexane and the resulting solvent was evaporated in vacuum at 60-70°C. Then purged with nitrogen and stored at -18°C.

The results are presented in table. 3, which shows the first and second peroxide values and the average of these two values, as well as the anisidine value (AnV). In FIG. 3 and 4 also show a decrease in POV and AnV (respectively for shorter and longer fermentations).

Table 3

Sample number Pasteurization temperature (°C) Pasteurization time (h) POV1 POV2 RO VcpeflH. AnV one - 0 6.0 5.4 5.7 25.7 2 40 one 8.8 8.5 8.6 25, 9 3 40 4 3.8 3.8 3.8 27.1 4 40 24 2.1 2.2 2.1 21.0 5 70 one 2.2 2.2 30.2 6 70 3.5 1.1 1.1 1.1 33.5 7 70 22 0.7 0.7 1.0 15, 9 eight 85 one 1.2 1.2 1.2 25, 9 nine 85 3.3 0.7 0.8 0.7 27.1 ten 85 20.5 0.5 0.5 0.5 12, 9 eleven - 0 5.9 5.4 5, 6 39.3 12 40 one 9.9 Yu,1 100 38, 7 thirteen 40 4 4.8 4.5 4.6 40.7 fourteen 40 24 2.5 h, o 2.8 32, 3 fifteen 70 one 2.7 2.8 2.7 40.3 sixteen 70 3.5 1.6 1.7 1.3 32.7 17 70 22 1.0 0.9 1.3 14.5 eighteen 85 one 1.8 1.8 1.8 39.7 nineteen 85 3.3 1.1 1.1 1.1 32, 4 20 85 20.5 0.9 1.0 0.9 16, 1

It follows from the data in the table that without pasteurization the POV was 5.6 or 5.7. Pasteurization at 40° C. reduced the POV, but it took a relatively long period of time (such as 24 hours) at the pasteurization temperature to bring the POV down to an acceptable value of 2.1.

Using higher temperatures resulted in a significantly better result. For example, when pasteurized for only 1 hour at 70°C, the POV value was 2.2 compared to a POV of 2.1 corresponding to 24 hours at 40°C. Even better results were obtained at higher temperatures, so at 85°C for 1 hour the POV value was only 1.2. (These figures are for shorter fermentations, although similar results were obtained with cells grown from longer fermentations.)

So, in Fig. 3 and 4 are graphical representations of POV and AnV values as a function of differences in pasteurization times. As expected, longer pasteurization resulted in lower AnV and POV values. However, the use of a relatively higher temperature during pasteurization was of great importance. A marked decrease in AnV and POV was found when the pasteurization temperature (T _paste ) was increased to 70°C, and even lower values corresponded to a temperature of 85°C. (The top three lines, marked with crosses, circles, and asterisks, show AnV values, while the bottom three lines, marked with diamonds, squares, and triangles, represent POV values.)

In table. 4 below shows the calculated tT product (in °C-min) for nine different pasteurization protocols (three different plateau temperatures and three different time periods). In fact, this product represents the area under the curve (dependence of time t, min on temperature T, ° C) for the plateau stage (after the heating stage, but before the cooling stage).

Table 4

Temperature (T, °C) 40 70 85 Time (t, h/min) 1(60) 2,400 4,200 5,100 4 (240) 9,600 16,800 20,400 24 (1440) 57,600 100,800 122.400

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Example 3

Subsequent pasteurization experiments were carried out using a fermentation broth, carrying out industrial scale fermentation using the M. alpina fungus exemplified above. Transported unpasteurized broth (800 l) and stored at 4°C. The broth was then transferred to a 700 L stirred tank and 10 different pasteurization protocols were performed.

First, pasteurization was carried out at five different (maximum) temperatures, namely 140, 120, 100, 80 and 60°C with a holding time (plateau) (at the maximum temperature) of 8 seconds. Second, pasteurization was carried out at 140, 120, 100, 80 and 60° C. with a holding time (plateau) at a maximum temperature of 300 seconds.

Samples were taken (2 l) and then immediately frozen at -18°C. Sterile samples (200 ml) were taken and frozen, and crude oil containing ARA was extracted from the samples.

A sample of the fermentation broth (1.7 l) was filtered under a N ₂ pressure of 1 bar. The precipitate was washed with 0.6 volumes of condensate water and squeezed out for about 5 minutes under a pressure of 400 kg/cm ² . n-hexane (500 ml) was then added to the wet cake and mixed using an Ultra Turrax at 24,000 rpm. The oil was extracted at room temperature (about 21° C.) for about 110 minutes. The suspension was vacuum filtered using Whatman GF/A media filter. The precipitate was washed with 250 ml of fresh hexane. Hexane was evaporated for 15 min on a water bath at a temperature of about 60-70°C. The resulting oil was then transferred into sealed beakers, which were purged with nitrogen for 30 seconds, and then sealed and stored at -18°C until analysis.

In FIG. Figures 5-7 show the data from the subsequent analysis. In FIG. 6 and 7 show time versus temperature curves for two series of runs, the first plateau (hold) time was 8 seconds and the second plateau (hold) time was 5 minutes, respectively, for 5 temperatures each. As can be seen from the graphs, the horizontal middle line (representing 8 s or 5 min) shows the plateau stage.

In FIG. Figure 5 shows the obtained POV and AnV values for all 10 pasteurization modes. As follows from these data, lower POV values were obtained at a significantly higher temperature, and a longer holding period (5 min) corresponded to the lowest POV values.

CLAIM

Claims (24)

CLAIM 1. A microbial oil that contains at least 35% polyunsaturated fatty acid (PUFA) and has an anisidine AnV value of no more than 25 and a POV value of no more than 3.0 obtained by pasteurization of microbial cells containing or producing PUFA, or microbial oil containing PUFA, where the pasteurization includes a first stage in which the microbial cells are heated from 40 to 70°C, and a second plateau stage in which the temperature is from 80 to 160°C. 2. An oil according to claim 1 wherein the PUFA is C ₂₀ or C ₂₂ PUFA. 3. The oil of claim 2 wherein the PUFA is arachidonic acid (ARA), eicosapentaenoic acid (EPA) or docosahexaenoic acid (DHA). 4. Oil according to any one of claims 1 to 3, which includes at least 40% PUFA. 5. Oil according to any one of claims 1 to 3, which includes at least 45% PUFA. 6. Oil according to any one of claims 1 to 5, which has an AnV of not more than 20. 7. Oil according to claim 6, where the oil has an AnV of not more than 15. 8. An oil according to any one of claims 1 to 7 which is extracted or isolated from cells which are fungi. 9. An oil according to claim 8, wherein the mushrooms belong to the genus Mortierella. 10. An oil according to claim 9, wherein the mushrooms are of the species Mortierella alpina. 11. The oil of claim 10 wherein the PUFA is arachidonic acid (ARA). 12. Oil according to claim 1, which is a crude or unrefined oil. 13. Oil according to claim 12, which includes at least 40% ARA. 14. Oil according to claim 12, which includes at least 45% PUFA. 15. Oil according to any one of claims 12-14, which has an AnV of not more than 20. 16. Oil according to claim 15, which has an AnV of not more than 15. 17. A human food product containing an oil according to any one of claims 1 to 16. 18. A nutritional supplement for humans comprising an oil according to any one of claims 1 to 16. 19. An animal feed containing an oil according to any one of claims 1 to 16. 20. An animal feed supplement containing an oil according to any one of claims 1 to 16. 21. An infant formula containing an oil according to any one of claims 1-16. 22. A method for refining a crude oil according to any one of claims 12 to 16, comprising subjecting the crude oil to one or more refining steps, wherein the refining steps include acid treatment or degumming, alkali treatment or removal of free fatty acid, bleaching - 11 039406 pigment removal or removal, filtration, fractionation by cooling, deodorization and/or clarification by filtration. 23. The method of claim 22 further comprising adding the resulting oil to human food or animal feed. 24. The method of claim 22, which further comprises adding the resulting oil to the infant formula.