COLOURING COMPOSITION FOR FOOD PRODUCTS

Abstract

The present invention relates to the use of an anthocyanin and/or betanin as a colourant in a food product, plant extracts as a source of anthocyanins and/or betanins, compositions comprising the plant extracts and processes using the anthocyanins and/or betanins or compositions described herein.

Claims

1. A process for colouring and controlling microbial growth in a food product selected from meat, fish or meat analogue product having about 1% or more fat by weight of the food product and/or has a pH of about 4 or more, wherein the process comprises the addition of i) one or more anthocyanin(s) obtained from red radish, ii) vinegar, and iii) rosemary extract to the food product.

2. The process according to claim 1, wherein the product is an emulsified product and the addition of one or more anthocyanin(s) to the food product is done before and/or during the emulsification process.

3. The process according to claim 2 further comprising a step of heating the food product.

4. The process according claim 1, wherein the anthocyanin is an anthocyanin glycoside.

5. The process according to claim 4, wherein the anthocyanin glycoside is acylated.

6. The process according to claim 1, wherein the food product has 5% or more by weight fat.

7. The process according to claim 1, wherein the vinegar is buffered.

8. The process according to claim 1, wherein the food product is processed meat, processed fish or processed meat analogue.

9. The process according to claim 1, wherein the food product is an emulsified food product.

10. The process according to claim 1, wherein the food product has been subjected to a temperature of 40° C. or above for at least 10 minutes.

11. The process according to claim 1, wherein the food product has an L* from about 60 to about 80, a* from about 10 to about 18, and b* from about 5 to about 15.

12. The process according to claim 1, wherein the controlling microbial growth comprises reducing or inhibiting the growth of Listeria, Clostridium and/or Salmonella.

13. The process according to claim 12, wherein the controlling microbial growth comprises reducing or inhibiting the growth of Clostridium botulinum.

14. A food colouring and anti-microbial composition comprising one or more anthocyanin(s) obtained from red radish, vinegar, and rosemary extract.

15. The composition of claim 14, wherein vinegar is present in an amount of about 30 to about 80 wt. %, the rosemary extract is present in an amount of about 2% to about 10%, and the one or more anthocyanin(s) is present in an amount of about 3% to about 20%, based on the total weight of the composition.

16. A food product containing the composition of claim 14 in an amount of about 100 ppm to about 10,000 ppm.

17. A food product obtained by the process of claim 1.

18. The food product according to claim 16, wherein the food product is selected from emulsified cooked sausage, cervelas or pates.

19. The food product according to claim 18, wherein the emulsified cooked sausage comprises frankfurter or hot dogs.

20. The food product according to claim 14, wherein the product is substantially devoid of nitrites and/or ascorbate.

Description

FIGURES

[0219] FIG. 1: Anthocyanin absorbance spectra.

[0220] FIG. 2: The L* value of sausages over time.

[0221] FIG. 3: The *a value of sausages over time.

[0222] FIG. 4: The b* value of sausages over time.

[0223] FIG. 5: Chromaticity diagram at d0 and d21.

[0224] FIG. 6: Hue angle overtime.

[0225] FIG. 7: Delta a* of sausages between d0 and d21.

[0226] FIG. 8: Delta E of sausages between d0 and d21.

[0227] FIG. 9: a* value at d0, comparison of process A and process B.

[0228] FIG. 10: a* value at d21, comparison of process A and process B.

[0229] FIG. 11: pH of all modalities.

[0230] FIG. 12: a* value during the shelf-life.

[0231] FIG. 13: Total plate counts in emulsified sausages during storage testing different conditions of blends

[0232] FIG. 14: Total plate counts in emulsified sausages during storage testing alternatives of buffered vinegar

[0233] FIG. 15: Lactic acid bacteria in emulsified sausages during storage testing alternatives of buffered vinegar

[0234] FIG. 16: Listeria growth during the shelf-life in emulsified sausages.

[0235] FIG. 17: Listeria growth during the shelf-life in fresh ground sausages.

[0236] FIG. 18: Growth differences between day 0 and day 4, and between day 4 and day 8 in fresh ground sausages

EXAMPLES

Material & Methods

[0237] 1. Plant Extracts

[0238] Red radish root (Raphanus sativus L.) was extracted with water and the liquid extract was dried on maltodextrin. The powder obtained was formulated on invert sugar syrup and water to obtain a standardized liquid extract with 4% of anthocyanins.

[0239] The juice of red beetroot (Beta vulgaris L.) was dried on maltodextrin to obtain a standardized powder extract with 0.3% of betanin.

[0240] Purple sweet potato root (Ipomoea batatas L.) was extracted with water and then purified by ethanol. The extract was dried on maltodextrin.

[0241] The blends were designed to ensure the antioxidant and antimicrobial functions of nitrite. These products are blends of three natural extracts: an extract of rosemary, a juice of acerola and vinegar.

[0242] See table 1 for proportion of each extracts in blends.

[0243] Carnosic acid+carnosol from the rosemary extract were present in the blend in an amount of about 1%, ascorbic acid from the acerola juice in an amount of about 4%, and acetic acid from vinegar in an amount of about 19%.

TABLE-US-00001 TABLE 1 Composition of blends tested Natstabil Natstabil Rosemary/ Rosemary Rosemary, Rosemary, M2 M2 Acerola FF/Acerola Acerola, Acerola, Qty % Blend 1 Blend 2 blend 1 blend 2 Pomegranate Citrus Buffered 68 68 — — — — vinegar Rosemary 2.6 — 2.6 2.6 2.6 additives 59% carnosic acid Rosemary — 13.4 — 13.4 — — Flavor 6% carnosic acid Acerola juice 9.4 9.4 9.4 9.4 9.4 9.4 powder (34% ascorbic acid) Carrier 20 9.2 88 77.2 53.1 18 Maltodextrin Citrus — — — — — 70 hesperidin (75%) Pomegranate — — — — 17 — extract (punicalagins > 4, ellagic acid 0.5-1%)

[0244] The rosemary aerial parts (historically named as Rosmarinus officinalis L., taxonomic change in Salvia rosmarinus Spenn.) were extracted with acetone at reflux. After the extraction was completed, the acetone extract was filtered to separate the solution from rosemary leaf and concentrated under reduced pressure to make concentrated native extract. At this time, the concentrated extract can be dried directly in a vacuum oven under mild heat to make a powdered extract, which is a composition comprising about 2%-10% carnosic acid and carnosol.

[0245] Alternatively, to the concentrated native extract, aqueous sodium carbonate (NaHCO.sub.3) was added to dissolve carnosic acid and other organic acids, while base insoluble substances were precipitated out.

[0246] The solution was filtered to separate from solid, and the filtrate was further concentrated under reduced pressure. Once finishing concentration is achieved, phosphoric acid (H.sub.3PO.sub.4) was added and the acid insoluble substances (including carnosic acid, carnosol, and carnosic derivatives) were precipitated from the concentrated solution. Charcoal active is used during the process to decolorize the rosemary extract in solution before filtration.

[0247] Through filtering, the precipitated solid was subsequently separated from liquid and rinsed with water to remove impurities.

[0248] Last, the solid was dried in a vacuum oven and then milled into powder to make a composition containing about 40-80% carnosic acid and carnosol.

[0249] Acerola cherry juice (Malpighia glabra L.) was dried to obtain an ascorbic acid-rich powder containing about 20-40% ascorbic acid.

[0250] Vinegar was dried to obtain an acetic acid-rich powder containing about 60-80% ascorbic acid.

[0251] 2. Colour and Stability Study in Solution

[0252] Red radish and purple sweet potato extracts were dissolved in buffer solutions pH 3 and pH 6. These solutions are storage over 21 days. The color was measured by L*a*b* on the first day and after 21 days.

[0253] L*a*b*

[0254] Chromametric analysis was performed by a chromameter Minolta CR100 in the color space CIE L*, a*, b* on the internal part of the meat product (sausage).

[0255] Color changes (L*a*b*) during storage were measured at day (d) 0, d8, d14 and d21.

[0256] 3. Emulsified Cooked Sausage Formulation and Processing

[0257] All the modalities are summarized in the Error! Reference source not found. below.

[0258] The extracts blends ensured the antioxidant and antimicrobial functions of nitrite, lactate and ascorbate/erythorbate. Red radish, beetroot and purple sweet potato extracts ensured the coloring function of nitrite and carmine.

TABLE-US-00002 TABLE 2 Modalities tested in emulsified cooked sausage  1 Negative control without nitrite, lactate, erythorbate, carmine  2 Positive control Nitrite/Erythorbate  3 3000 ppm Natstabil M2 blend1 + 4000 ppm Red Radish  4 3000 ppm Natstabil M2 blend1 + 4000 ppm Red Radish + 1500 ppm Beetroot  5 3000 ppm Natstabil M2 blend1 + 4000 ppm Purple sweet potato  6 3000 ppm Natstabil M2 blend1 + 4000 ppm Beetroot  7 3000 ppm Natstabil M2 blend1 + 1000 ppm Red raddish + 1000 ppm Beetroot + 500 ppm Flavor  8 3000 ppm Natstabil M2 blend1 + 1000 ppm Red raddish + 1000 ppm Beetroot  9 3000 ppm Natstabil M2 blend1 10 3000 ppm Rosemary/Acerola blend1 11 3000 ppm Rosemary FF/Acerola blend2 12 3000 ppm Rosemary, Acerola, Pomegranate 13 4000 ppm Rosemary, Acerola, Citrus 14 3000 ppm Rosemary, Acerola Blend 1 + vinegar 8%

[0259] The emulsified cooked sausage ingredients are summarized in the Error! Reference source not found. below.

TABLE-US-00003 TABLE 3 Emulsified cooked sausage ingredients Pork meat % 56% Pork fat % 24% Water + Ice % 20% Salt g/kg 18 White Pepper g/kg 2 Garlic powder g/kg 1 Nutmeg powder g/kg 0.5 Coriander powder g/kg 1 Dextrose g/kg 5 Lactose g/kg 5 Plasma g/kg 10 Polyphosphates g/kg 5 Erythorbate g/kg 0.5 Nitrite salt g/kg 18 Extract Blends according to table 2

[0260] For each modality, 8 kg of sausage were produced. Two emulsion processes were tested for each modality.

[0261] During the Process A, pork meat and salt were ground firstly, then polyphosphates were added. In the next step, water and ice were added and mixed with the ground meat. To emulsify the product, pork fat was then added and ground. Finally, spices, natural extracts, sugars and plasma were incorporate in this emulsion.

[0262] During the Process B, pork meat and salt were grounded firstly, then polyphosphates and natural extracts were added. In the next step, water and ice were added, then pork fat, spices, sugars and plasma and the product was emulsified.

[0263] After the emulsion process, the mix was embossed in a VISCOFAN skinless cellulose casing (21 mm diameter).

[0264] Sausages were then cooked at 99% of relative humidity at 50° C. for 20 min, 55° C. for 10 min, 74° C. for ring 23 min and finally cooled to 10° C. for 5 min (10% relative humidity).

[0265] A part was inoculated on surface with Listeria monocytogenes for the challenge test and the other part was kept safe for shelf life microbiological study as well as organoleptic and physical characterization. Cooked emulsified sausages were then stored in vacuum packaging.

[0266] 4. Fresh Sausage Formulation and Processing

[0267] All the modalities are summarized in the Error! Reference source not found. below.

[0268] The extracts Blend 1 and Blend 2 ensured the antioxidant and antimicrobial functions of nitrite, lactate and ascorbate/erythorbate.

TABLE-US-00004 TABLE 4 Modalities tested in fresh sausage 1 Control without nitrite, lactate, erythorbate 2a Nitrite/Erythorbate 2b Lactate/Erythorbate 3 3000 ppm NatStabil M2 blend 1 4 4000 ppm NatStabil M2 blend 1 5 2000 ppm NatStabil M2 blend 2 6 3000 ppm NatStabil M2 blend 2

[0269] Fresh sausage ingredients are summarized in the Error! Reference source not found. below.

TABLE-US-00005 TABLE 5 Fresh sausage ingredients Pork meat 4D % 86% Pork fat % 14% Salt g/kg 18 Pepper g/kg 1 Garlic powder g/kg 0.3 Dextrose g/kg 4 Lactose g/kg 1 Water + Ice g/kg 55 Erythorbate g/kg 0.5 Nitrite salt g/kg 18 Lactate g/kg 25 Extract Blends according to table 4

[0270] For each modality, 6 kg of ground pork were produced.

[0271] Pork meat was ground with fat and with all the other ingredients.

[0272] A part of the ground meat was inoculated with Listeria monocytogenes for the challenge test, homogenized and embossed. The other part was kept safe and embossed for organoleptic and physical characterization. Fresh ground sausages were then conditioned in a modified atmosphere packaging (MAP) containing 80% O.sub.2 and 20% CO.sub.2.

[0273] 5. Microbiological Analysis

[0274] Microbiological analysis was performed according to current standard EC regulation 2073/2005, at d0 after packaging, several days during the storage (from D4 to D7 or D14 according to the studies).

[0275] For fresh ground sausage, 10 days of shelf-life with 4 days of storage at 4° C. and 6 days at 8° C., to simulate a break in the cold chain (standardized method NF V01-003)

[0276] For cooked emulsified sausage, 14 days of shelf-life with 7 days of storage at 4° C. and 7 days at 8° C., to simulate a break in the cold chain (standardized method NF V01-003).

[0277] Group of microorganisms counts performed are listed below: [0278] Total plate count (NF EN ISO 4833-1), [0279] Lactic acid bacteria (NF ISO 15214) both mesophilic and psychotrophic [0280] Enterobacteriaceace (ISO 21528-2), [0281] Pseudomonas sp. (EN ISO 13720), [0282] Brochothrix thermosphacta [0283] Anaerobic enumeration of sulfito-reducing bacteria (NF V08-061), [0284] Challenge test Listeria monocytogenes (NF-EN ISO 11290-2). [0285] Challenge test Salmonella (NF-EN ISO 6579-1).

[0286] Logarithmic values of Listeria growth (log CFU/g) were calculated for each experiment and treatment.

[0287] Differences of logarithmic values of Listeria growth (log CFU/g) between the meat treated with plant extracts (or nitrite or lactate) and the untreated control were calculated to yield a final result. The more negative value was obtained, the higher was the antilisterial effect of the extract or of the combination of extracts. In meat science microbiology, for a given time, values are considered to be significant between two series when a difference of 0.5 Log 10 CFU/g is observed (Chaillou et al., 2014); (Guide pour la validation de methodes d'essais microbiologiques et revaluation de leur incertitude de mesure dans les domaines de la microbiologie alimentaire et de l'environnement), Schweizerische Eidgenossenschaft, Confederation Suisse, Departement federal de l'economie, de la formation et de la recherche DEFR, Document No. 328, April 2013, Rev. 03). In microbiology, it will be noted that a treatment has a significant antibacterial effect if its effect exceeds −0.5 log CFU/g as compared to the untreated control.

[0288] Results & Discussion

[0289] 1.1 Color & stability Study in Solution

[0290] In buffered solution, red radish and purple sweet potato are bright red and stable at pH 3. However, at pH 6, red radish and purple sweet potato become purple.

[0291] After 21 days of storage at pH 6, the purple color is degraded in pale brownish purple, with ΔE (color difference between after and before storage) at 7.37 and 13.84 respectively, which represent important color differences.

[0292] 1.2 Color & Stability Study in Emulsified Sausage

[0293] Before cooking, the control meat was light pink (1) and meat with nitrite was grey (2) (usual shades). With red radish extract (3), beetroot extract (6) and mixture thereof (4), the meat had a slightly more intense pink shade than control. With purple sweet potato (5), meat was intense purple.

[0294] Dispersibility of all extracts was correct and meat color was homogenous. Moreover, extracts adding did not influence meat pH (Error! Reference source not found. and FIG. 11).

[0295] It is surprising that red radish modalities revealed pink shades and not purple shades at the pH found in meat. These results are not in line with the previous study in buffer solutions and the bibliographic data. The difference between red radish and purple sweet potato could be due to the composition of the anthocyanins. Major anthocyanins in both extracts are acylated but purple sweet potato is richer in acylated peonidin glycosides and acylated cyaniding glycosides, while red radish is richer in acylated pelargonidin glycosides.

TABLE-US-00006 TABLE 6 pH of all modalities before and after cooking pH before pH after pH pH pH Modalities cooking cooking d0 d7 d14 d21 1- Control 6.28 6.42 6.41 6.45 6.44 2- Nitrite 6.31 6.48 6.43 6.47 6.47 3- Red Radish 6.27 6.47 6.41 6.45 6.46 4- Red Radish + Beetroot 6.25 6.43 6.41 6.45 6.46 5- Purple Sweet Potato 6.23 — 6.41 6.45 6.44 6- Beetroot 6.27 — — 6.46 6.41

[0296] After cooking, the control was grey (1) and sausage with nitrite was light pink (2) (usual shades). With the red radish extract (3) and mixture (4), only a slight loss of color was observed. This loss gave a colour similar to the nitrite control (FIG. 2). With the beetroot extract (6), sausage was light pink close to colour provided by nitrite but with an orange shade. With the purple sweet potato extract (5), the sausage was pale purple. The extracts did not influence meat pH (Error! Reference source not found.).

[0297] It is surprising that red radish modalities displayed the exact nitrite-like pink shade after cooking; this is not in line with the previous study in buffer solutions and the bibliographic data.

[0298] After 21 days of storage (10 days at 4° C. and 11 days at 8° C.), the control was still grey (1) and shade of sausage with nitrite (2) seemed to be slightly degraded (pale orange shade).

[0299] The sausages prepared with red radish extract (3) and the mixture (4) were surprisingly still pink; the color seemed to be more stable over time than nitrite sausage color.

[0300] The shade of the sausage with the beetroot extract (6) was degraded in pale orange and sausage with purple sweet potato extract (5) was still purple.

[0301] The extracts did not influence meat pH (Error! Reference source not found.).

[0302] This last result is also surprising because the color was stable during the 21 days of storage although the sausage pH was above 6. This unusual stability could be due to the acylation of anthocyanins, however, the literature showed that acylated anthocyanins were not stable at pH 7.

[0303] These visual observations can be confirmed by L*a*b* measurements.

[0304] L* value, the lightness from black (0) to white (100), was stable over time for all modalities (FIG. 2).

[0305] Regarding a* value (from green (−) to red (+)), control showed weak a* values, stable over time, which confirm the normal grey color observed (FIG. 3). Nitrite modality presented a* values more important than control, stable over time, which confirm normal pink color observed.

[0306] Modalities with red radish (3) and mixture (4) showed a* values very close to nitrite modality and stable over time. Furthermore, purple sweet potato modality (5) displayed the most important a* values, stable over time. However, beetroot modality (6) showed an important a* value at d0 but this value was not stable over time. This indicates that pink color was not stable and confirm the visual observations and the bibliographic data.

[0307] Concerning b* value (from blue (−) to yellow (+)), control was stable over time (FIG. 4).

[0308] Nitrite modality showed a slight increase of b* value at d21, which confirm the slight color degradation observed (orange shade). Modalities with red radish (3) and mixture (4) showed b* values very close to nitrite modality, slightly lower and even more stable over time, which confirms the stable and intense pink color observed. However, purple sweet potato modality (5) displayed a negative b* value, which indicate a blue shade and confirm the purple color observed. Furthermore, beetroot modality (6) showed an increase of b* value over time, which indicates that pink color is degraded in orange and confirms the visual observations and the bibliographic data.

[0309] In order to illustrate L*a*b* measurement in two dimensions, FIG. 5 expose a part of the chromaticity diagram for all modalities at d0 and d21. It is obvious that control, nitrite, red radish, blend and purple sweet potato modalities had a stable shade over time, with values at d0 and d21 very close. However, for beetroot alone, there was an important shift between d0 and d21 values. This observation confirms the instability of the beetroot pink color.

[0310] In summary, the results and figures confirm the surprising results that the combination of red radish+beetroot extracts provide a good colour shade, and also provide high colour stability, that are better than nitrite, and also better than would have been predicted from the colour and stability of the extracts on their own. This demonstrates a synergistic effect. The results and figure also show that although the purple sweet potato extract did not provide the require colour, the colour was highly stable.

[0311] Combining Hue Angle (FIG. 6), delta a* (FIG. 7) and delta E (FIG. 8) calculations, we can confirm L*a*b* observations.

[0312] The Hue Angle gives a numerical estimate of the color of the sausages. The hue sequence on a CIELAB diagram is defined with red-purple (0°), yellow (90°), bluish-green (180°) and blue (270°).

[0313] The hue angle may be defined as the angle between the hypotenuse and 0° on the a* axis; h is calculated from the arctangent of b*/a*. Arctangent, however, assumes positive values in the first and third and negative values in the second and fourth quadrants. For a useful interpretation, h° should remain positive between 0° and 360° of the color sheet. h ° is calculated following the equation below:

h.sub.ab°=degrees(arctangent(b*/a*))

The control had an important hue angle value (≈70°), which confirms the pale yellow shade (FIG. 6).

[0314] Nitrite, red radish extract, the mixture of red radish extract and beetroot juice, and the beetroot juice had intermediate values (≈25-50°), which confirms the pink shade of all these modalities. It is important to notice that the beetroot juice hue angle increased during time, which confirms the lack of stability of this product alone.

[0315] At the contrary, red radish extract and the mixture of red radish extract and beetroot juice had a perfectly stable hue angle, more pink than beetroot alone and nitrite. Concerning the mixture of red radish extract and beetroot juice, the calculation of expected values (added in the FIG. 6) showed that expected color was more orange and less stable than obtained color.

[0316] This difference in Hue Angle reveal a synergistic behavior between red radish extract and beetroot juice and could be attributed to interaction of betanin with anthocyanins or other unknown compounds (intermolecular co-pigmentation).

[0317] This synergistic result is also visible with delta a* (Δa*) calculation (FIG. 7) and delta E (ΔE) calculation (FIG. 8).

[0318] Delta a* gives the difference of a* between day 0 and day 21 following the equation below:

Δa*=a*.sub.d21−a*.sub.d0

Where a*.sub.d21 is a* value at day 21 and a*.sub.d0 is a* value at day 0.

[0319] Delta E (ΔE) gives the difference of L*, a* and b* between day 0 and day 21 following the equation below:

ΔE=√{square root over ((L*.sub.21d−L*.sub.0d)+(a*.sub.21d−a*.sub.0d)+(b*.sub.1d−b*.sub.0d))}

[0320] Where L*a*b*.sub.21d are the coordinates at day 21 and L*a*b*.sub.0d are the coordinates at day 0.

[0321] The Control modality, the Nitrite modality, the red radish extract, the mixture of red radish extract and beetroot juice, and the purple sweet potato extract were stable during time with a Δa*<1. However the beetroot juice alone displayed a Δa*=−6.05, showing an important colour degradation (red decreasing). Thus, the mixture of red radish extract and beetroot juice should be less stable than observed, the calculation of expected value indicated a Δa*=−1.42 whereas the observed value was only −0.46. This difference of 1 point confirmed the synergistic behavior between red radish extract and beetroot juice.

[0322] In the same manner, red radish extract showed a ΔE=3.94 and beetroot juice showed a ΔE=8.96. The calculation of expected value of the mixture of red radish extract and beetroot juice indicated a ΔE=2.61 whereas the observed value was only 1.26. Here again, this difference of more than 1 point confirmed the synergistic behavior between red radish extract and beetroot juice.

[0323] Further organoleptic studies were done. It was shown that the combination of red radish extract and beetroot juice when compared to a standard reference, do not modify the smell and taste of the final product (such as hot dogs or fresh sausages).

[0324] 1.3 Comparison of Process a and Process B of Emulsified Sausages

[0325] Both emulsification processes were performed and final color of cooked sausage was measured by L*a*b* at d0 and d21 (FIG. 9 and FIG. 10).

[0326] Concerning the control and the nitrite modalities, a* values were equivalent in processes A and B.

[0327] However, a* values with natural extracts were substantially more important in process B in 25 comparison of process A.

[0328] This difference is surprising and showed a stabilization effect of the incorporation at an early stage of the process.

[0329] 1.4 Color and Stability Study of Fresh Ground Sausage

[0330] Dispersibility of all extracts was homogenous during process. Moreover, extracts adding did not influence initial meat pH and pH overtime (FIG. 10).

[0331] Fresh ground sausages showed a good red color preservation overtime, except for the control. This observation was confirmed by a* measurements in FIG. 12, there was no significant loss of red between the modalities, but a tendency for the negative control with lower a* values.

[0332] These results revealed that Natstabil M2 Blend 1 and Natstabil M2 Blend 2 were as effective as lactate/ascorbate to protect meat color during shelflife.

[0333] 2.1 Microbiological Preservation of Cooked Emulsified Sausage

[0334] 2.1.1. Spoilage

[0335] The microbiological analysis showed that the total flora and the lactic flora had a normal growth for the nitrite and the lactate modalities and for the natural extracts modalities, moreover, the ASR, the Enterobacteria and the Pseudomonas were absent (<10 ufc/g). However, the control displayed the highest growth of total flora, Enterobacteria and Pseudomonas.

[0336] The microbiological analysis showed equivalent results between control, nitrite and natural extracts.

[0337] Main known group of bacteria found in meat product were targeted during the 14 days of storage. The total plate counts are shown in FIGS. 13 and 14. Normal growth is observed, with a diminution of the load after cooking step and then an increasing of total plate counts during the shelf life. After 14 days, emulsified sausages with blends have shown a lower level of contamination (total plate counts) than the negative control and positive control (with Nitrites).

[0338] It is remarkable that the blend with Natstabil M2 and red radish and beetroot have even a better antimicrobial effect than Natstabil M2 alone (see FIG. 13).

[0339] FIG. 15 shows similar results for psychrotophic lactic acid bacterias.

[0340] 2.1.2. Pathogens

[0341] The challenge test with Listeria displayed a low growth under 4° C., but at 8° C. (day 14 & 21) the growth is important for all the modalities, this meat product is sensible and nitrite cannot limit the growth as the natural extracts (FIG. 16).

[0342] Statistically, negative control is significantly different to the others and natural extracts are equivalent to nitrite.

[0343] 2.2 Microbiological Preservation of Fresh Ground Sausage

[0344] The challenge test with Listeria (FIG. 17 and FIG. 18) confirmed that nitrite has no action against Listeria in this application (Δ log cfu/g<−0.5 at d4 and d8), only lactate can limit its growth (Δ log cfu/g=−0.9 at d8).

[0345] Same limitation of Listeria growth was observed for Natstabil M2 Blend 1 and Natstabil M2 Blend 2 at the both dosages, with values not significantly different to lactate control (Δ log cfu/g>−1 at d8) (FIG. 17 and FIG. 18) These results revealed that Natstabil M2 Blend 1 and Natstabil M2 Blend 2 were as effective as lactate to protect meat from Listeria.

[0346] Organoleptic studies were done. It was shown that the both blends are equivalents regarding the taste and the smell if compared with a standard reference. Thus the Blends 1 and Blend 2 do not modify the smell and taste of the final product (such as hot dogs or fresh sausages).

[0347] Conclusion

[0348] Surprisingly, the mixture of red radish extract and beetroot juice is pink and stable after cooking and during storage, more stable than expected with calculations.

[0349] Purple sweet potato has a too purple shade to replace nitrite in this sausage application.

[0350] Red Radish and Red Radish in mixture with Beetroot could be alternatives to nitrite and even more stable based on the colour stability investigations undertaken.

[0351] Anti-Clostridium Study

[0352] In the present study, the behavior of Clostridium botulinum (group II type B) during the process and the storage of cooked emulsified sausages stored vacuum-packed using different formulations (including a nitrite replacer) was evaluated.

[0353] 1. Materials and Methods

[0354] 1.1. Spores of C. botulinum Preparation

[0355] A cocktail of three strains of C. botulinum type B non-proteolytic and toxin producer (BL7; 300.05 and 815.12 from Pasteur institute, France) were used.

[0356] After a culture step in BHI media 30° C. for 12 weeks under anaerobic conditions, spores were able to germinate, vegetative cells were able to grow and sporulation was possible.

[0357] After that, cells were exposed to a heated treatment at 60° C. for 20 min to destroy and vegetative cells and keep only the spores. Spores were centrifuged at 4500×g for 20 min, washed and put in a buffer solution.

[0358] 1.2. Emulsified Cooked Sausage Model and Validation

[0359] The method was based on the following published procedure from Redondo-Solano et al. (2013) to study Clostridium perfringens risks in cooked ham. It has been extrapolated to a mix of minced pork meat. The meat mix was inoculated with the bacteria of interest, packed under vacuum packed in 50 g portions. Regarding the risks of C. botulinum biosafety reason, the embossing step was not able to be performed and the mixed meat have been stored under vacuum packed instead.

[0360] The emulsified cooked sausages ingredients were summarized in Table 1.

TABLE-US-00007 TABLE 1 Emulsified cooked sausages ingredients. Negative Positive Ingredients control Assay 1 Assay 2 control Pork meat (kg) 3.4 3.4 3.4 3.4 Pork fat (kg) 1.4 1.4 1.4 1.4 Water + Ice (kg) 1.2 1.2 1.2 1.2 Salt NaCl (g) 0 108 108 0 White Pepper (g) 0 12 12 12 Garlic powder (g) 0 6 6 6 Nutmeg powder (g) 0 3 3 3 Coriander powder (g) 0 6 6 6 Dextrose (g) 30 30 30 30 Plasma (g) 0 60 60 60 Lactose (g) 0 30 30 30 Polyphosphates (g) 0 0 0 0 Sodium erythorbate (g) 3 0 0 3 Nitrite salt (g) 0 0 0 108 Extract blend 1 (g) 0 18 0 0 Extract blend 2 (g) 0 0 18 0 Total (kg) 6.033 6.303 6.303 6.288

[0361] For each modality, pork meat and salt were ground firstly, then polyphosphates and natural extracts were added. In the next step, water and ice were added and mixed with the ground meat. To emulsify the product, pork fat was then added and ground. Finally, spices, sugars and plasma were incorporated in this emulsion.

[0362] The ground meat with C. botulinum spores at 102-103 spores per gram for the challenge test. Homogenization was performed using a kitchen blender (Kenwood Major Titanium, Kenwood, Japon) speed 1 for 10 minutes.

[0363] After the emulsion process, the mix was not embossed due to safety reasons (inoculation of C. botulinum). Cooked emulsified sausages were then stored in vacuum packaging using equipment (INV 10, Intervac, France) and specific bags (Cryovac® CN 300, Sealed Air, France). Samples were prepared in triplicates.

[0364] Sausages were then cooked at 77° C. for 155 min and finally cooled down from 65° C. to 8° C. in 210 min, and from 8° C. to 4° C. in 60 min.

[0365] Sausages were stored under controlled temperatures during 40 days shelf life at 4° C. for 14 days followed by a break down in cold chain at 20° C. for 2 hours. After that, samples were kept at 8° C. for 26 days.

[0366] 1.3. Extract Blends Composition

[0367] The extract's blend ensured the antioxidant and antimicrobial functions of nitrite, lactate and ascorbate/erythorbate.

[0368] Blend 1: buffered vinegar 68% (with a content of acetic acid of about 25%), acerola juice powder 9.4% (with a content of ascorbic acid of 34%), Rosemary extract 2.6% (59% carnosic acid).

[0369] Blend 2: Rosemary Flavor 13.4% (6% carnosic acid), acerola juice powder 9.4% (with a content of ascorbic acid of 34%).

[0370] 1.4. Physico-Chemical Properties Evaluation

[0371] Water activity (aw) and pH were determined at day 0 just after the cooking and cooling down step, and at day 40 on samples from the same batches but without inoculation of C. botulinum spores.

[0372] For water activity, NF ISO 18787 (AFNOR, 2017) was followed using aw-meter from Aqualab 4TE, Meter Group, Allemagne. Measures were performed at 25° C.

[0373] For pH, ISO 2917: 1999 procedure was followed using Mettler-Toledo LoT406-M6-DXK-S7/25 (Urdorf, Suisse).

[0374] 1.5. Enumeration of C. botulinum

[0375] After incubation of the suspension at 60° C. for 20 min, enumeration of C. botulinum vegetative cells and spores was performed on classical culture media (TS media 30° C. in anaerobic conditions for 24 to 48 h) on the day 0, 21, 30 and 40.

[0376] In parallel, lactic acid bacteria were enumerated on MRS agar after incubation at 30° C. for 24 h to 48 h under anaerobic conditions at day 0 after cooking step and day 40.

[0377] Total plate counts were evaluated on PCA after incubation at 30° C. for 24 h to 48 h under anaerobic conditions at day 0 before and after cooking step and day 40.

[0378] 1.6. Detection of Botulinum Toxin by Bio-Assay

[0379] Botulinum toxin detection was performed by bio-assay on mice (5 samples for modalities) at day 21 and day 40.

[0380] Detection of toxin is performed injecting the knack suspension to mice. In order to be compliant with ethics regulation, when a mouse died due to toxin presence, the other mice were not injected, and the analysis was stopped.

[0381] 2. Results

[0382] 2.1. Physico-Chemical Properties Evaluation

[0383] Results of water activity and pH of emulsified cooked sausages are shown table 2.

TABLE-US-00008 TABLE 2 Water activity and pH of cooked emulsified sausage model samples inoculated and stored 14 days at 4° C., 2 h at 20° C. and 26 days at 8° C. aw pH D0 after D0 after cooking D40 cooking D40 Blend 1 0.99 0.97 6.30 6.10 0.98 0.99 6.25 6.20 0.99 0.98 6.20 6.25 Blend 2 0.99 0.99 6.15 6.15 0.99 0.98 6.15 6.15 0.98 0.98 6.15 6.20 Positive control 0.98 0.99 6.15 6.20 with nitrite 0.98 0.98 6.15 6.20 0.98 0.98 6.15 6.10 Negative control 1.00 1.00 6.20 6.20 without nitrite 1.00 1.00 6.20 6.20 1.00 1.00 6.20 6.20

[0384] Results were compliant with physicochemical properties commonly found in cooked emulsified sausages.

[0385] 2.1 C. botulinum Enumeration

[0386] Enumerations of C. botulinum by microbiological cultural approach are shown for assessing the level of inoculation and followed during the storage, respectively for both spores and vegetative cells in table 3 and for only spores (after heating treatment 20 min at 60° C.) in table 4.

TABLE-US-00009 TABLE 3 C. botulinum (spores + vegetative cells) inoculated in cooked emulsified sausage model samples inoculated and stored 14 days at 4° C., 2 h at 20° C. and 26 days at 8° C. in CFU/g. D0 before D0 after cooking cooking Day 21 Day 30 Day 40 Blend 1 500 35 <5 15 <5 750 25 <5 20 5 600 20 <5 65 5 Blend 2 850 <5 5 5 <5 700 <5 15 <5 35 1000 45 20 <5 15 Positive control 250 5 15 <5 5 with nitrite 450 20 10 <5 5 400 25 30 5 <5 Negative control 1600 <5 <5 <5 10 without nitrite 1200 <5 <5 <5 20 900 <5 <5 <5 2000

[0387] Nitrite are known to inhibit the growth of vegetative cells of C. botulinum, and the germination of the spores. Similar to the positive control, meat model made using blend 1 and 2 have shown this inhibition compare to one sample without nitrite.

[0388] In order to check if the detection was vegetative cells or spores, a heated treatment is made to kill the vegetative cells. Enumeration is performed afterwards to detect the spore only. Results of level of spores only are shown table 4.

TABLE-US-00010 TABLE 4 C. botulinum (spores only) inoculated in cooked emulsified sausage model samples inoculated and stored 14 days at 4° C., 2 h at 20° C. and 26 days at 8° C. in CFU/g. D0 after cooking Day 21 Day 30 Day 40 Blend 1 10 <5 5 <5 <5 <5 <5 <5 10 <5 <5 <5 Blend 2 <5 <5 <5 <5 <5 <5 <5 <5 5 <5 <5 <5 Positive control <5 <5 <5 <5 with nitrite <5 <5 <5 <5 5 <5 <5 <5 Negative control 5 <5 <5 <5 without nitrite <5 <5 <5 <5 5 <5 <5 <5

[0389] Similar than controls, they were below detection level of C. botulinum spores at the end of the shelf life in meat model with blend 1 and 2.

[0390] Level of lactic acid bacteria were also performed and shown table 5. This results it is shown as control of the potential competition of the inoculated bacteria.

TABLE-US-00011 TABLE 5 Endogenous lactic acid bacteria in cooked emulsified sausage model samples inoculated and stored 14 days at 4° C., 2 h at 20° C. and 26 days at 8° C. in CFU/g. Lactic acid bacteria D0 after cooking Day 40 Blend 1 5 <5 <5 <5 <5 <5 Blend 2 <5 <5 <5 <5 <5 <5 Positive control with nitrite <5 <5 <5 <5 <5 <5 Negative control without nitrite <5 <5 <5 <5 <5 <5

[0391] 2.2. Toxin Detection

[0392] In order to detect the production of toxin, sample of cooked emulsified sausage model samples inoculated were put in suspension to be inoculated to mice. Detection of toxin was done at day 21 and day 40 and results are shown Table 6.

TABLE-US-00012 TABLE 6 Detection of toxins of C. botulinum on mice bio-assays in 5 cooked emulsified sausage model samples inoculated and stored 14 days at 4° C., 2 h at 20° C. and 26 days at 8º C. D0 after cooking Day 21 Day 30 Day 40 Blend 1 ND 0/5 ND 0/5 Blend 2 ND 0/5 ND 1/5* Positive control with nitrite ND 0/5 ND 0/5 Negative control without 0/5 0/5 ND 3/5** nitrite *1 positive sample **3 positive samples ND non detected

[0393] In the table 6, positive sample means that at least one mouse is dead after meat suspension injection. When 0/5 result occur that mean, mouse injected with the meat suspension is alive, no toxin production is detected.

[0394] It is known in the literature (Majou D et al.) that the use of nitrite can inhibit the production of C. botulinum toxin and put the cured meat product at risk for consumer. The results in the negative control without the use of nitrite is confirming this statement. Toxins were produced by C. botulinum at the end of the shelf life when no preservative agent were used. We demonstrate that using the blend 1 solution (buffered vinegar, acerola, rosemary extract) or positive control (nitrites) we can inhibit the production of toxin, and keep the meat product safe for the consumer.

REFERENCES

[0395] Sharma, Shashi & Whiting, Richard. (2005). Methods for Detection of Clostridium botulinum Toxin in Foods. Journal of food protection. 68. 1256-63. 10.4315/0362-028X-68.6.1256. [0396] Majou D, Christieans S. Mechanisms of the bactericidal effects of nitrate and nitrite in cured meats. Meat Sci. 2018 November; 145:273-284. doi: 10.1016/j.meatsci.2018.06.013. Epub 2018 Jul. 10. PMID: 30005374