Improved single-cell protein production using antioxidants

Abstract

The invention relates to a method for preparing a stable composition of single-cell protein by using antioxidants. The single-cell protein can be derived from a fungal biomass, wherein the biomass is pasteurised after antioxidants have been added to it. The stable composition has low TOTOX values and is suitable for use as a source of single-cell proteins in food or feed products. The invention also relates to the pasteurised product, and to food products comprising it.

Claims

1. Method for producing a biomass composition comprising an antioxidant and biomass, comprising the steps of: i) providing a biomass; ii) adding an antioxidant to the biomass to obtain a biomass mixture; iii) pasteurising the biomass mixture to obtain the biomass composition; and iv) optionally washing the biomass composition.

2. The method according to claim 1, wherein the antioxidant is at least one of: a) a free radical scavenger, such as carnosol; carnosic acid; rosemary extract; a flavan-3-ol such as epicatechin, epigallocatechin, epigallocatechin gallate (EGCG), epicatechin gallate; a flavonoid such as kaemferol, quercetin, or myricetin; green tea extract; butylated antioxidants such as butylated hydroxytoluene (BHT), butylated hydroxyanisole, or tert-butylhydroquinone (TBHQ); gallic acid; a gallate ester such as propyl gallate; carvone; a caffeate ester such as methyl caffeate, ethyl caffeate, caffeic acid phenethyl ester (CAPE), or rosmarinic acid; spearmint extract; a vitamin E such as a tocopherol or a tocotrienol; a vitamin A such as retinol, retinoic acid, or retinal; a provitamin A such as a carotenoid, β-carotene, lutein, zeaxanthin, or echinenone; a quinolone-based antioxidant such as ethoxyquin; or seaweed extract; or b) an oxygen reducer, such as ascorbic acid or acerola extract; or c) a chelator, such as citric acid or EDTA.

3. The method according to claim 1, wherein the obtained biomass composition has a TOTOX value of under 50, and wherein the TOTOX value preferably remains under 50, for at least 6 weeks.

4. The method according to claim 1, wherein the biomass is derived from a fermentation broth, wherein the fermentation broth preferably comprises a biomass dry matter content of at most 6%, more preferably of at most 4%, or wherein the fermentation broth is sieved to obtain the biomass.

5. The method according to claim 4, wherein the biomass comprises at most 500 mg/kg dry matter iron.

6. The method according to claim 1, wherein the pH of the biomass in step iii) is at most 4.5.

7. The method according to claim 1, wherein the biomass comprises at least 7% dry matter.

8. The method according to claim 1, wherein the biomass is a biomass derived from a fungal strain, wherein the fungal strain is preferably a strain of a fungal genus selected from the group consisting of Rasamsonia, Talaromyces, Penicillium, Acremonium, Humicola, Paecilomyces, Chaetomium, Rhizomucor, Rhizopus, Thermomyces, Myceliophthora, Thermoascus, Thielavia, Mucor, Stibella, Melanocarpus, Malbranchea, Dactylomyces, Canariomyces, Scytalidium, Myriococcum, Corynascus, and Coonemeria.

9. The method according to claim 1, wherein the pasteurisation is performed in an in-line heating unit that preferably comprises a pipe heater, a heating block, or a steam infusion element, and wherein the in-line heating unit optionally comprises a mixing element such as a static mixer.

10. The method according to claim 1, wherein the pasteurisation is performed for at most 45 minutes.

11. The method according to claim 1, wherein pasteurisation is performed at a temperature of at least 70° C.

12. The method according to claim 1, wherein antioxidant is only added during step ii).

13. Biomass composition comprising an antioxidant and biomass, obtained by a method as described in claim 1.

14. Food or feed product comprising the biomass composition according to claim 13.

15. (canceled)

16. The biomass composition according to claim 13, wherein the biomass comprises from about 6% to 12% lipids and from about 35% to 55% proteins, based on dry weight.

17. The method according to claim 8, wherein the fungal genus is Rhizomucor.

18. The method according to claim 8, wherein the fungal the strain is Rhizomucor pusillus.

19. The method according to claim 18, wherein the Rhizomucor pusillus strain is CBS 143028, or a strain that is a single colony isolate or a derivative thereof.

Description

DESCRIPTION OF DRAWINGS

[0151] FIG. 1A-7D kill curve for pasteurisation of fungal biomass comprising 10% dry matter.

[0152] FIG. 1B-12D kill curve for pasteurisation of fungal biomass comprising 10% dry matter.

EXAMPLES

Example 1—Production of Pasteurised Single-Cell Protein

[0153] Rhizomucor pusillus strain CBS 143028 was fermented as described in WO2018/029353. Alternately, Rhizomucor pusillus strain CBS 143028 is inoculated in 200 ml of a preculture medium at pH 5.5 containing a defined mineral composition containing KCl 0.5 gr/L; KH.sub.2PO.sub.4 4, Na.sub.2HPO.sub.4 1.1, Citric acid 1.5 gr/L, MgSO.sub.4.7 aq 2 gr/L, FeSO.sub.4.7 aq 0.1 gr/L, CaCl.sub.2.2 aq 0.1 gr/L, ZnSO.sub.4.7 aq 0.125 gr/L, MnCl.sub.2.4 aq 0.012, CuSO.sub.4.5 aq 0.0016 gr/L, CoCl.sub.2.6 aq 0.0015 gr/L, Na2B.sub.4O.sub.7.10 aq 0.009 gr/L KI 0.0009 gr/L, Na.sub.2MoO.sub.4.2 aq 0.0015 gr/L; 11 g Dextrose per l as C-source; 4 g (NH.sub.4).sub.2SO.sub.4 per l as N-source; and 7.5 g tartaric acid per l. The culture is incubated for 24 hours at 46° C., in a 1 l Erlenmeyer flask with air permeable stop with baffles, in an orbital shaker at 200 rpm. The preculture is then used to inoculate a pH 3.5 medium containing a defined mineral medium as described above comprising 77 g Dextrose per l as C-source; 1.4 g (NH.sub.4).sub.2SO.sub.4 per l as N-source and supplemented with NH3 as titrant. Olive oil is continuously being fed. More definitions are described in EP20153414.

[0154] Fermentation broths, having reached a dry matter content ranging from 2 to 5 weight percent, were concentrated using a vibrating sieve to achieve a minimum of 10% (wt.) dry matter. The biomass is then pumped through an in-line heating unit comprising a pipe equipped with a steam injector. Pumping speed, pipe diameter, and temperature were configured in such a way that different residence times at different temperatures were achieved. Systematic screening of parameters allowed the generation of killing curves for various degrees of log.sub.10 reduction, notably for a log.sub.10 reduction of 7 (see FIG. 1A), and for a log.sub.10 reduction of 12 (see FIG. 1B). Data is shown in the table below. Pasteurised samples were conveniently dried in a fluid bed drier (70° C. 10 min).

TABLE-US-00001 TABLE 1.1 kill kinetics using an in-line pasteurisation unit, showing required time in minutes T (° C.) 7 D 8 D 9 D 10 D 11 D 12 D 60 2280.53 2606.32 2932.11 3257.91 3583.70 3909.49 70 79.27 90.60 101.92 113.25 124.57 135.90 71 57.27 65.45 73.63 81.81 89.99 98.17 72 41.45 47.37 53.29 59.21 65.13 71.05 73 30.05 34.35 38.64 42.93 47.23 51.52 74 21.83 24.95 28.07 31.19 34.31 37.43 75 15.89 18.16 20.43 22.70 24.97 27.24 76 11.59 13.24 14.90 16.55 18.21 19.86 77 8.46 9.67 10.88 12.09 13.30 14.51 78 6.19 7.08 7.96 8.85 9.73 10.62 79 4.54 5.19 5.84 6.48 7.13 7.78 80 3.33 3.81 4.29 4.76 5.24 5.71 81 2.45 2.80 3.15 3.50 3.85 4.20 82 1.81 2.07 2.32 2.58 2.84 3.10 83 1.33 1.52 1.71 1.91 2.10 2.29 84 0.99 1.13 1.27 1.41 1.55 1.69 85 0.73 0.83 0.94 1.04 1.15 1.25 86 0.54 0.62 0.70 0.77 0.85 0.93 87 0.40 0.46 0.52 0.58 0.63 0.69 88 0.30 0.34 0.39 0.43 0.47 0.51 89 0.22 0.26 0.29 0.32 0.35 0.38 90 0.17 0.19 0.21 0.24 0.26 0.29

Example 2—Organoleptic Qualities of Pasteurised Single-Cell Protein

[0155] Pasteurised biomass obtained as described above was subjected to analysis by a testing panel. Sample 1 was pasteurised at 74° C. for 37 minutes. Sample 2 was pasteurised at 86° C. for 1 minute. Sample 3 was not pasteurised.

[0156] A testing panel of 12 people reported the following scent sensations for sample 1: metallic, ferric, oxidised, French fries, sunflower oil, raw egg. Samples 2 and 3 were generally described as odourless. Mean marks awarded for scent were 51% for sample 1, and 86% for samples 2 and 3.

Example 3—Determination of TOTOX Values for Pasteurised Single-Cell Protein

[0157] The TOTOX value is an indication for the degree of oxidation and can be described by the following equation: (TOTOX=2PV+AV). Herein PV represents the peroxide value and AV the anisidine value. Assays for these values are known in the art. In this example, AV was determined following NEN-EN-ISO 6885:2000, PV by the certified service provider Nutrilab B.V. (Giessen, the Netherlands). Pasteurised SCP was prepared as described in Example 1, where 1000 ppm antioxidant was added at different points of the process: [0158] A) Mixing of the antioxidant after sieving and before pasteurisation and washing. [0159] B) Mixing of the antioxidant after sieving, pasteurisation, and washing. [0160] C) Mixing half of the total amount of antioxidant before pasteurisation and half after washing. [0161] D) Mixing of antioxidant after washing and before pasteurisation.

[0162] For each strategy two different antioxidants were tested, one water soluble (a mixture of carnosol and carnosic acid (rosemary extract) and flavan-3-ols (green tea extract), further comprising excipients E1520 (propylene glycol), E471 (mono- and diglycerides of edible fatty acids), and E433 (polyoxyethylene sorbitan monooleate); this mixture is available as NaturFORT 12L from Kemin) and one oil soluble (a mixture of carnosol and carnosic acid (rosemary extract) and flavan-3-ols (green tea extract), further comprising excipients E1520, E471, and E322 (Lecithin); this mixture is available as NaturFORT 15L from Kemin). Results are shown in Table 2. Before the analysis of the samples they were dried in a fluid bed dryer at 50° C. for 20 minutes and incubated at 60° C. for 3 days, mimicking a shelf life study to amplify the difference in oxidation as part of it will take place during storage.

TABLE-US-00002 TABLE 2 results of different antioxidant (AOX) addition strategies Solubility Strategy of AOX PV AV TOTOX A Water 9.4 21.0 39.8 A Oil 7.8 15.7 31.3 B Water 8.4 21.6 38.4 B Oil 7.5 17.0 32.0 C Water 7.5 24.0 39.0 C Oil 6.8 36.5 50.1 D Water 6.5 18.9 31.9 D Oil 4.7 25.9 35.3 No AOX — 9.7 19.2 38.6
Oil-soluble antioxidants lead to better TOTOX values.

Example 4—Determination of TOTOX Values after Prolonged Storage

[0163] The strategy of adding the antioxidant prior to pasteurisation and washing was used for this follow-up study, where antioxidants were used at 2000 ppm. SCP was freeze dried after production, and AV, PV, and TOTOX values were determined either immediately thereafter, or after storage at 55° C. for six weeks (which represents storage at 20° C. for 1.3 years). Values were determined for SCP derived from biomass that was produced in broths with varying iron contents (by using broths with varying amounts of iron). The sample using a broth with 350 mg/kg (dry matter) iron was produced by slow growing biomass, the other three values were produced by fast growing biomass. Table 3 shows the results.

TABLE-US-00003 TABLE 3 TOTOX values for various conditions after prolonged storage iron content (mg/kg T = 0 6 weeks at 55° C. dry matter) PV AV TOTOX PV AV TOTOX 350 17.7 3.9 39.3 14.7 11.9 41.3 130 4.6 5.6 14.8 36.9 9 54.9  47.3 5.5 2.2 13.2 14 3.5 31.5  41.2 5.4 1.8 12.6 7.2 5.9 20.3
Reduced iron content of the pasteurised biomass leads to improved TOTOX values.