Fruit ferments containing propionate and use thereof

Abstract

Methods to prepare a fruit ferment containing propionic acid and/or a salt thereof comprising i) obtaining a liquid fruit preparation, ii) optionally supplementing the liquid fruit preparation with water and/or with additional components to support fermentation, iii) fermenting the optionally supplemented liquid fruit preparation with a propionic acid-producing bacterial strain, iv) optionally further processing the fermentation product, to obtain the fruit ferment. Next to a propionic acid-producing bacterial strain, a lactic acid-producing bacterial strain may be used in the fermentation. And, a fruit ferment obtainable by said method.

Claims

1. A fruit ferment obtained by fermentation of a liquid fruit preparation with a propionic acid-producing bacterial strain, said fermentation comprising the steps of: i) obtaining a liquid fruit preparation having a sugar level of from 1.5 to 10% (w/w) and an initial pH of from 4.5 to 7; ii) fermenting the liquid fruit preparation with a propionic acid-producing bacterial strain whilst maintaining the pH between 4.5 and 7 by the addition of a base selected from the group consisting of sodium hydroxide, magnesium hydroxide, calcium hydroxide, ammonium hydroxide, calcium carbonate and ammonium carbonate; and iii) optionally further processing the fermentation product, to obtain the fruit ferment, wherein the propionic acid-producing bacterial strain used in said fermenting is supplemented with one or more strains of lactic acid producing bacteria, wherein said fruit of the fruit ferment does not include nuts and cereal grains, wherein said fruit ferment comprises: propionate in a concentration ranging from 0.25 to 80% (w/w); and succinate in a concentration ranging from 0.002 to 1% (w/w), wherein the weight ratio of propionate to succinate is in the range from 200 to 15, wherein the propionate does not include potassium propionate.

2. The fruit ferment according to claim 1 further comprising acetate in a concentration ranging from 0.1 to 60% (w/w), the weight ratio of propionate to acetate ranging from 0.5 to 10.

3. The fruit ferment according to claim 1, wherein the weight ratio of propionate to succinate is in the range from 150 to 25.

4. The fruit ferment of claim 2, wherein the weight ratio of propionate to succinate is in the range from 150 to 25.

5. The fruit ferment according to claim 1 containing part or all of the propionic acid-producing bacterial cells and the lactic acid-producing bacterial cells of the fermentation.

6. The fruit ferment according to claim 1, wherein said fermenting is substantially devoid of microorganisms other than said propionic acid-producing bacterial strain and said one or more strains of lactic acid producing bacteria.

7. A composition comprising a fruit ferment and additional additive ingredients for food and/or beverages, wherein said fruit of the fruit ferment does not include nuts and cereal grains and wherein said fruit ferment comprises: propionate in a concentration ranging from 0.25 to 80% (w/w); and succinate in a concentration ranging from 0.002 to 1% (w/w), wherein the weight ratio of propionate to succinate is in the range from 200 to 15, wherein the propionate does not include potassium propionate, wherein said fruit ferment is obtained by fermentation of a liquid fruit preparation with a propionic acid-producing bacterial strain, said fermentation comprising the steps of: i) obtaining a liquid fruit preparation having a sugar level of from 1.5 to 10% (w/w) and an initial pH of from 4.5 to 7; ii) fermenting the liquid fruit preparation with a propionic acid-producing bacterial strain whilst maintaining the pH between 4.5 and 7 by the addition of a base selected from the group consisting of sodium hydroxide, magnesium hydroxide, calcium hydroxide, ammonium hydroxide, calcium carbonate and ammonium carbonate; and iii) optionally further processing the fermentation product, to obtain the fruit ferment, wherein the propionic acid-producing bacterial strain used in said fermenting is supplemented with one or more strains of lactic acid producing bacteria.

8. A food or beverage comprising a fruit ferment, wherein said fruit of the fruit ferment does not include nuts and cereal grains and wherein said fruit ferment comprises: propionate in a concentration ranging from 0.25 to 80% (w/w); and succinate in a concentration ranging from 0.002 to 1% (w/w), wherein the weight ratio of propionate to succinate is in the range from 200 to 15, wherein the propionate does not include potassium propionate, wherein said fruit ferment is obtained by fermentation of a liquid fruit preparation with a propionic acid-producing bacterial strain, said fermentation comprising the steps of: i) obtaining a liquid fruit preparation having a sugar level of from 1.5 to 10% (w/w) and an initial pH of from 4.5 to 7; ii) fermenting the liquid fruit preparation with a propionic acid-producing bacterial strain whilst maintaining the pH between 4.5 and 7 by the addition of a base selected from the group consisting of sodium hydroxide, magnesium hydroxide, calcium hydroxide, ammonium hydroxide, calcium carbonate and ammonium carbonate; and iii) optionally further processing the fermentation product, to obtain the fruit ferment, wherein the propionic acid-producing bacterial strain used in said fermenting is supplemented with one or more strains of lactic acid producing bacteria.

9. The food or beverage according to claim 8 further comprising the same fruit used in preparing the fruit ferment.

10. A probiotic consisting of a fruit ferment obtained by fermentation of a liquid fruit preparation with a propionic acid-producing bacterial strain, said fermentation comprising the steps of: i) obtaining a liquid fruit preparation having a sugar level of from 1.5 to 10% (w/w) and an initial pH of from 4.5 to 7; ii) fermenting the liquid fruit preparation with a propionic acid-producing bacterial strain whilst maintaining the pH between 4.5 and 7 by the addition of a base selected from the group consisting of sodium hydroxide, magnesium hydroxide, calcium hydroxide, ammonium hydroxide, calcium carbonate and ammonium carbonate; and iii) optionally further processing the fermentation product, to obtain the fruit ferment, wherein the propionic acid-producing bacterial strain used in said fermenting is supplemented with one or more strains of lactic acid producing bacteria, wherein said fruit of the fruit ferment does not include nuts and cereal grains; wherein said fruit ferment comprises: propionate in a concentration ranging from 0.25 to 80% (w/w); and succinate in a concentration ranging from 0.002 to 1% (w/w), wherein the weight ratio of propionate to succinate is in the range from 200 to 15, wherein the propionate does not include potassium propionate, and wherein said fruit ferment contains part or all of the propionic acid-producing bacterial cells and the lactic acid-producing bacterial cells of the fermentation.

11. A fruit ferment comprising: propionate in a concentration ranging from 0.25 to 80% (w/w); and succinate in a concentration ranging from 0.002 to 1% (w/w), wherein the weight ratio of propionate to succinate is in the range from 200 to 15, wherein the propionate does not include potassium propionate, and wherein said fruit of the fruit ferment does not include nuts and cereal grains.

12. The fruit ferment according to claim 11 further comprising acetate in a concentration ranging from 0.1 to 60% (w/w), the weight ratio of propionate to acetate ranging from 0.5 to 10.

13. The fruit ferment according to f claim 11, wherein the weight ratio of propionate to succinate is in the range from 150 to 25.

14. The fruit ferment according to claim 12, wherein the weight ratio of propionate to succinate is in the range from 150 to 25.

Description

EXAMPLE 1

(1) Preparation of Various Fruit Ferments

(2) P. freudenreichii was precultured at a starting pH of 6.5, on a medium containing 15 g/l Difco certified yeast extract and 30 ml/1 of a 50% sodium lactate solution. The culture was incubated at 30? C. until it was fully grown. Lactobacillus paracasei was precultured on MRS medium (de Man et al. 1960, J Appl Bact 23 (130-135), A Medium for the Cultivation of Lactobacilli), purchased from Oxoid, at a start pH of 5.8.

(3) Several cases were tested, with different media compositions. Each case is listed below in Table 1, wherein the amount of puree or juice per liter fermentation medium is indicated:

(4) TABLE-US-00001 Ingredients per liter fermentation medium Case (adjusted to 1 liter with water) 1 210 g Tomato paste (providing 35.7 g/L sugar) 2 0.5 L water melon juice (freshly prepared) 3 0.5 L water melon juice (freshly prepared) 10 g difco yeast extract 4 0.75 L watermelon juice (freshly prepared) 5 0.25 L apple juice (freshly prepared) 6 0.33 L orange juice (freshly prepared) 7 0.25 L white grape juice (freshly prepared) 8 0.33 L pineapple juice (freshly prepared)

(5) The juices in Table 1 were prepared by a standard juice extractor, in this case a Philips HR1858 juicer. After obtaining the juice, it was immediately put into a fermentation reactor without further filtration.

(6) Fermentation reactors were sterilized at 121? C. for 20 minutes. The pH in the reactor was maintained at pH 6.5 with 2.5 M NaOH (100 g/L). Stirring of the media was done at 250 rpm.

(7) At the start of a fermentation, 10% v/v of a culture of lactic acid bacteria and 10% v/v of a culture of propionic acid bacteria were added to diluted juice.

(8) After fermentation (48-96 hours), solids including biomass were removed using centrifugation at 7000?g for 20 minutes.

(9) TABLE-US-00002 TABLE 1 Acids present in juice after fermentation and solids removal (case 4 not determined) Case Case Case Case Case Case Case Case component 1 2 3 4 5 6 7 8 formic 0.12 0.12 0.19 n.d. 0.04 0.12 0.03 0.07 acid acetic 0.86 0.6 0.8 n.d. 0.52 0.7 0.60 0.71 acid propionic 1.5 1.5 1.4 n.d. 1.2 1.1 1.4 1.4 acid ethanol <0.02 0.02 0.04 n.d. <0.01 0.03 <0.01 <0.01 butyric <0.01 <0.01 <0.01 n.d. <0.01 <0.01 <0.01 <0.01 acid pyruvic <0.02 <0.02 <0.02 n.d. <0.02 <0.02 <0.02 0.02 acid lactic <0.05 <0.05 <0.05 n.d. <0.05 <0.05 <0.05 <0.05 acid 2-hydroxy <0.01 <0.01 <0.01 n.d. <0.01 <0.01 <0.01 <0.01 butyric acid oxalic <0.02 <0.01 <0.01 n.d. <0.01 <0.01 <0.01 <0.01 acid sorbic <0.01 <0.01 <0.01 n.d. <0.01 <0.01 <0.01 <0.01 acid fumaric <0.02 <0.01 <0.01 n.d. <0.01 <0.01 <0.01 <0.01 acid succinic 0.10 0.02 0.01 n.d. 0.03 0.02 0.04 0.05 acid benzoic <0.03 <0.03 <0.03 n.d. <0.03 <0.03 <0.03 <0.03 acid maleic <0.02 <0.01 <0.01 n.d. <0.01 <0.01 <0.01 <0.01 acid

(10) After solids removal, the ferments were concentrated 8-16 times by rotary vacuum evaporation, operating at 80 mbar. The organic acid composition of the concentrated ferments was determined (Table 2):

(11) TABLE-US-00003 TABLE 2 Concentration of acids after vacuum evaporation (case 1 not determined) Case Case Case Case Case Case Case Case component 1 2 3 4 5 6 7 8 formic n.d. 0.51 1.0 1.4 0.24 1.0 0.13 0.37 acid acetic 8.6 3.0 4.1 9.9 5.4 6.5 4.7 4.3 acid propionic 15 6.8 7.9 22 14 11 12 8.8 acid ethanol n.d. <0.01 <0.01 <0.01 <0.01 <0.01 <0.01 <0.01 butyric n.d. <0.01 <0.01 <0.01 <0.01 <0.01 <0.01 <0.01 acid pyruvic n.d. <0.02 <0.02 <0.02 <0.02 <0.02 <0.02 <0.01 acid lactic n.d. <0.05 <0.05 0.07 <0.05 <0.05 <0.05 <0.05 acid 2-hydroxy n.d. <0.01 <0.01 0.01 <0.01 <0.01 <0.01 <0.01 butyric acid oxalic n.d. <0.01 <0.01 <0.01 <0.01 0.01 <0.01 0.01 acid sorbic n.d. <0.01 <0.01 <0.01 <0.01 0.02 <0.01 0.02 acid fumaric n.d. <0.01 <0.01 <0.01 <0.01 <0.01 <0.01 <0.01 acid succinic n.d. 0.09 0.04 1.2 <0.01 0.9 1.0 0.6 acid benzoic n.d. <0.03 <0.03 <0.03 <0.03 <0.03 <0.03 <0.03 acid maleic n.d. <0.01 <0.01 <0.01 <0.01 <0.01 <0.01 <0.01 acid

(12) In the same manner as described above, four ferments (cases 9, 10, 11 and 12) were prepared, but now using a fermentation medium containing per liter of water: 25 g/l Difco yeast extract and 70 g/l sucrose. After biomass removal and concentration, case 9 contained 22% (w/w) propionic acid and case 10 contained 20.4% (w/w) of propionic acid (other acids were not determined). In case 11, the ferment was evaporated to dryness using a rotary evaporator at reduced pressure (60-100 mbar). In case 12, the ferment of case 9 was taken and sodium lactic acid was added until the ferment contained 25% (w/w) of lactic acid. After the addition of lactic acid, the ferment contained 13.7% (w/w) of propionic acid.

EXAMPLE 2

(13) Effect of Tomato Ferment on Yeasts in Tomato Sauce

(14) The efficacy of a tomato ferment was tested in tomato sauce. Tomato sauce was prepared having a composition according to Table 3:

(15) TABLE-US-00004 TABLE 3 Composition of tomato sauce Ingredient Dosage (%) Description Supplier Tomato puree 30.0 Double concentrated 28/30%; Perfekt ingredients: tomatoes and salt Salt 3.2 Table salt Jozo Sugar 20.0 Cyrstalline sugar CSM Water 45.54 Vinegar 1.26 (=0.36% 100%) (28.5%) TOTAL 100 pH = 3.8 (adjusted with HCl or NaOH), aW = approximately 0.94

(16) A propionate-containing sucrose ferment was prepared based on sucrose and yeast extract, containing 22% propionic acid (case 9 of Example 1). Additionally a concentrated tomato ferment was prepared containing 15% propionic acid (case 1 of Example 1). These ferments were added to batches of tomato sauce according to Table 4.

(17) The following yeast strains were cultivated on GPY broth (5 g/l yeast extract, 4 g/l glucose, 5 g/l peptone, at a pH of 5-5.5), and added to the tomato sauce, according to table 4, at an end concentration of approximately 1000 cells per gram: Pichia membranaefaciens MUCL 27794, Candida tropicalis MUCL 28180, Zygosaccharomyces rouxii MUCL 30008. The tomato sauce was incubated at 20? C. with the indicated yeast strain and the indicated ferment (Table 4). Regularly, the colony forming units (cfu) were determined using MEA plates (20 g/l malt extract, 20 g/l glucose, 1 g/l peptone, 20 g/l agar), incubated at 20? C.

(18) TABLE-US-00005 TABLE 4 Test set up of a microbiological experiment with sucrose ferment (case 9) and tomato ferment (case 1) in tomato sauce Tomato Sucrose ferment ferment Log cfu Log cfu added added at day at day Sample Name (% w/w) (% w/w) Microorganism 7 80 1 control 0 0 Pichia membranaefaciens 3 9.5 1 control 0 0 Candida tropicalis 1.5 <1 1 control 0 0 Zygosaccharomycesrouxii 4 8.5 2 Low tom 1 0 Pichia membranaefaciens <1 <1 2 Low tom 1 0 Candida tropicalis <1 <1 3 High tom 3 0 Pichia membranaefaciens <1 <1 3 High tom 3 0 Candida tropicalis <1 <1 4 Low prop 0 0.6 Pichia membranaefaciens <1 <1 4 Low prop 0 0.6 Candida tropicalis 1.5 <1 5 High prop 0 1.8 Pichia membranaefaciens <1 <1 5 High prop 0 1.8 Candida tropicalis <1 <1 6 Med tom 1.7 0 Zygosaccharomycesrouxii 1.5 <1 7 Med prop 0 1 Zygosaccharomycesrouxii 1 <1

(19) The tomato juice based ferment was as effective as the sucrose based ferment for at least 80 days. It was clear from the results that Candida tropicalis was not able to grow on tomato sauce as the colony forming units went down in the control experiment in the first 7 days.

EXAMPLE 3

(20) Taste of Melon Ferments Compared to Other Propionate Ferments

(21) This experiment provides a description of the taste of these ferments in application and a comparison on taste with other propionate ferments. The comparison is based on the ISO 8587:2006 sensory analysis ranking test. The taste of the ferments is evaluated in a model drink. The recipe of this model drink is presented in Table 5.

(22) TABLE-US-00006 TABLE 5 recipe of model drink Ingredient Supplier Amount [g] Water 949.85 Sucrose Granulated sugar-Van Gilse 40.0 Apple juice Cargill 8.30 concentrate Apple flavour Givaudan 55078-DO 0.35 Citric acid Across, M&A-021 1.50

(23) In this model drink the following ferments are compared to each other: PSP9 Liquid (see case 10 of Example 1): Concentrated liquid propionic acid ferment based on sucrose and yeast extract with a propionic acid content of 20.4% (w/w). PSP9 Powder (see case 11 of Example 1): Powder of the ferment described above with a propionic acid content of 41% (w/w). Melon ferment with yeast extract: Propionic ferment based on watermelon juice+10 g/L yeast extract, concentrated. Melon ferment without yeast extract: Propionic ferment based on watermelon juice, concentrated. PQ (see case 12 of Example 1): Propionic acid ferment based on sucrose and yeast extract, mixed with lactic acid to a total of 25%.

(24) The ferments were compared at a propionate level of 0.05%. Table 6 shows the propionate level of each ferment and the amount that should be added to the model drink to reach a level of 0.05%.

(25) TABLE-US-00007 TABLE 6 propionate levels and dosage levels of the ferments Ferment Propionate level [%] Dosage level [%] PSP9 Liquid 20.4 0.245 PSP9 Powder 41.0 0.122 Melon ferment with 7.9 0.633 yeast extract Melon ferment with- 6.8 0.735 out yeast extract PQ 13.7 0.365

(26) All samples were tasted by 8 trained panelists. The panelists were asked to rank the samples from least to most different from the reference sample. The reference sample is pure model drink.

(27) Table 7 shows the pH of the samples and the description of the taste.

(28) TABLE-US-00008 TABLE 7 pH and description of the taste of samples with different propionate ferment Sample pH Description X (reference = 3.18 Fresh, apple model drink) PSP9 Liquid 4.21 Less fresh and apple flavour, strong ferment taste PSP9 Powder 4.23 Less fresh and apple flavour, little ferment taste Melon ferment with 4.30 Less fresh and apple flavour, sweet yeast extract Melon ferment with- 4.24 Less fresh and apple flavour, sweet, out yeast extract little bitter PQ 3.92 Less fresh and apple flavour, sour
Ranking

(29) The samples were ranked from least to most different from the reference. Table 8 shows the results of the ranking.

(30) TABLE-US-00009 TABLE 8 ranking of 5 propionate ferments by 8 panellists Panellist PSP9 L PSP9 P Melon + Y Melon ? Y PQ 1 5 3 1 2 4 2 5 3 2 1 4 3 5 4 1 2 3 4 5 4 2 1 3 5 5 2 1 3 4 6 4 1 3 2 5 7 5 3 1 2 4 8 5 1 4 2 3 Sum 39 21 15 15 30 SD 0.35 1.19 1.13 0.64 0.71 1 = least different from reference x, 5 = most different from x n = 8 k = 5

(31) For a panel size of 8 panelists and 5 products to rank the total score needs to be lower than 17 and or higher than 31 to indicate a significant difference (one-tailed test). It can be seen that both the samples with melon ferment with and without yeast extract have significant less influence on taste compared to the other samples. The sample with PSP9 Liquid has significant more influence on taste than the other samples. The conclusion is that melon ferments have less influence on the taste of apple juice model drink than other propionate ferments. The melon ferments have also a different taste profile; they are sweeter than the other ferments.

EXAMPLE 4

(32) Fermentation of Honey Melon and Apple Juice with Propionibacterium

(33) Preparation of the Preculture

(34) One vial (1 ml) with a ?80? C. glycerol stock of Propionibacterium freudenreichii ss. shermanii was inoculated in flask of 100 ml containing a medium containing 20 g/l of glucose and 15 g/l of yeast extract and incubated at 30? C. for 2 days. The 100 ml of culture was transferred to a 1 L bottle containing 900 ml of the same medium (20 g/l of glucose and 15 g/l of yeast extract). This bottle was incubated for 1 day at 30? C.

(35) Fermentation of Fruit Juice

(36) A 1 L fermenter was prepared with a diluted commercial melon juice concentrate. The concentrate was diluted to a sugar concentration of approximately 45 g/l, and 1% of yeast extract paste (50% paste from Biospringer) was added and subsequently the reactor including medium was autoclaved. The fermentation was started by adding 100 ml of the Propionibacterium preculture. A similar reactor was filled with a diluted commercial apple juice concentrate. The final sugar concentration was also 45 g/l. Both reactors were kept at 30? C., stirred at 150 rpm and pH controlled at a pH of 6.5 using an ADI1020 controller and 5M NaOH as a base for pH correction. The two reactors were sampled regularly and the sugar concentration and propionic acid concentration were measured. The results of these fermentations are presented in tables 8 and 9. Although the formation of propionic acid was relatively slow, the glucose was rapidly converted (<48 h) to propionic acid. This suggests that fruits with high glucose content are optimal in these kinds of fermentations.

(37) TABLE-US-00010 TABLE 9 concentrations of acids and sugars in the Honeydew Melon juice fermentation (g/l) Propionic Acetic Succinic Hour acid acid acid Glucose Fructose Sucrose 0 0 0.2 0 13.5 18.1 15.4 24 2.4 1.4 0.4 10.8 16.4 14.5 48 6.2 3.2 0.8 <1 14.7 14.5 72 7.4 4.1 1.5 <1 11.8 14.1 100 7.9 4.4 2.0 <1 9.7 14.1 118 8.2 4.6 2.4 <1 8.0 13.1

(38) TABLE-US-00011 TABLE 10 concentrations of acids and sugars in the Apple juice fermentation (g/l) Propionic Acetic Succinic Hour acid acid acid Glucose Fructose Sucrose 0 0 0.1 0 15.3 27.5 2.5 24 1.3 1.0 0.6 12.4 27.5 2.4 48 5.4 3.1 1.5 <1 24.5 2.4 72 6.3 3.7 2.4 <1 21.0 2.4 100 7.1 4.2 2.8 <1 18.1 2.4 118 7.9 4.6 3.3 <1 15.7 2.5