Storage stable frozen lactic acid bacteria culture

Abstract

A storage stable frozen lactic acid bacteria (LAB) culture that comprises LAB that are that are able to utilize sucrose, has a weight of at least 50 g frozen material and a content of viable bacteria of at least 10.sup.9 colony forming units (CFU) per g frozen material.

Claims

1. A frozen lactic acid bacteria culture comprising: a mixture of mesophilic lactic acid bacteria that (a) have an optimum growth temperature of about 30° C. and (b) are capable of utilizing sucrose, and from 0.5% to 80% w/w of at least one cryoprotective agent, wherein the frozen culture is not freeze-dried and has a weight of at least 50 grams and comprises at least 10.sup.9 colony forming units of viable bacteria per gram.

2. The frozen culture of claim 1, wherein the culture is a LD-culture comprising Lactococcus lactis subsp. lactis, Lactococcus lactis subsp. cremoris, Lactococcus lactis subsp. lactis biovar. diacetylactis and Leuconostoc mesenteroides subsp. cremoris.

3. The frozen culture of claim 1, wherein the culture is an L-culture.

4. The frozen culture of claim 1, further comprising a thermophilic Streptococcus thermophilus bacterium.

5. The frozen culture of claim 1, further comprising a thermophilic Lactobacillus acidophilus bacterium.

6. The frozen culture of claim 1, wherein the at least one cryoprotective agent comprises a carbohydrate.

7. The frozen culture of claim 1, wherein the at least one cryoprotective agent comprises a disaccharide.

8. The frozen culture of claim 1, wherein the at least one cryoprotective agent comprises at least one of trehalose and sucrose.

9. The frozen culture of claim 1, wherein the at least one cryoprotective agent comprises a mixture of a disaccharide and a polysaccharide.

10. The frozen culture of claim 9, wherein the disaccharide comprises sucrose and the polysaccharide comprises maltodextrin.

11. The frozen culture of claim 1, wherein the at least one cryoprotective agent is selected from the group consisting of a protein, a protein hydrolysate, and an amino acid.

12. The frozen culture of claim 1, wherein the frozen culture comprises from 3% to 50% w/w of the at least one cryoprotective agent.

13. The frozen culture of claim 1, wherein the frozen culture comprises from 4% to 10% w/w of the at least one cryoprotective agent.

14. A food or feed product made using the frozen culture of claim 1.

15. A method of making a food or feed product, wherein the method comprises adding the frozen culture of claim 1 to a food or feed material.

16. A method for making a frozen lactic acid bacteria culture comprising: (i) adding at least one cryoprotective agent to a mixture of mesophilic lactic acid bacteria that (a) have an optimum growth temperature of about 30° C. and (b) are capable of utilizing sucrose; and then (ii) freezing the culture, wherein the frozen culture is not freeze-dried and has a weight of at least 50 grams and comprises (a) at least 10.sup.9 colony forming units viable bacteria per gram and (b) from 0.5% to 80% w/w of the at least one cryoprotective agent.

17. The method of claim 16, wherein the culture is a LD-culture comprising Lactococcus lactis subsp. lactis, Lactococcus lactis subsp. cremoris, Lactococcus lactis subsp. lactis biovar. diacetylactis and Leuconostoc mesenteroides subsp. cremoris.

18. The method of claim 16, wherein the frozen mixture comprises from 3% to 50% w/w of the at least one cryoprotective agent.

19. The method of claim 16, wherein the frozen culture comprises from 4% to 10% w/w of the at least one cryoprotective agent.

Description

DRAWINGS

(1) FIGS. 1 to 4: Stability profiles for F-DVS of F1 DaN & CH N11. For further details see working Example 1.

(2) FIGS. 5 to 10: Stability profiles for F-DVS of F1 DaN, CH N11 & CH N19. For further details see working Example 2.

(3) FIG. 11: Stability profiles for R603. For further details see working Example 3.

DETAILED DESCRIPTION OF THE INVENTION

(4) A Frozen Lactic Acid Bacteria (LAB) Culture

(5) The term “mixed lactic acid bacteria (LAB) culture” denotes a mixed culture that comprises two or more different LAB species. The term a “pure lactic acid bacteria (LAB) culture” denotes a pure culture that comprises only a single LAB species specie.

(6) The culture as described herein may be a mesophilic culture consisting of mesophilic bacteria having optimum growth temperatures at about 30° C.

(7) The culture as described herein comprises LAB that are that are able to utilize sucrose. The Leuconostoc mesenteroides subsp. cremoris is able to utilize sucrose. Among others, it is present in a L-culture and a LD-culture.

(8) Consequently, in a preferred embodiment the frozen culture is a L-culture or more preferably a LD-culture. A L-culture and a LD-culture are examples of mesophilic cultures. Further they are mixed cultures. Consequently, a culture as described herein is preferably a mixed culture, more preferably a mesophilic mixed culture.

(9) A L-culture comprises Lactococcus lactis subsp. lactis, Lactococcus lactis subsp. cremoris and Leuconostoc mesenteroides subsp. cremoris.

(10) A LD-culture comprises Lactococcus lactis subsp. lactis, Lactococcus lactis subsp. cremoris, Lactococcus lactis subsp. lactis biovar. diacetylactis and Leuconostoc mesenteroides subsp. cremoris.

(11) The specific amount of the individual bacterial species may vary in accordance with the specific required use. The skilled person is aware of this and capable of determining the preferred mixed culture composition according to the required needs.

(12) For instance, if aroma is required a relatively high percentage of the aroma making bacteria Lactococcus lactis subsp. lactis biovar. diacetylactis and Leuconostoc mesenteroides subsp. cremoris could be preferred.

(13) A preferred LD-culture comprises:

(14) TABLE-US-00001 Lactococcus lactis subsp. lactis, 60-95%, Lactococcus lactis subsp. cremoris preferably 70-90% Lactococcus lactis subsp. lactis biovar. 5-40%, diacetylactis, Leuconostoc mesenteroides preferably 10 to 30% subsp. cremoris

(15) Within the ranges above, it is preferred to have from 0.25 to 6% of Leuconostoc mesenteroides subsp. cremoris and from 7 to 30% of Lactococcus lactis subsp. lactis biovar. diacetylactis.

(16) Of course the total percentage sum of the 4 different LAB specifies cannot exceed 100%. However, it may be less than 100% if other bacteria than the 4 mentioned ones are present in the LD-culture. Working examples 1 and 2 herein provides examples of stabilized LD-cultures.

(17) The culture as described herein may be a thermophilic culture consisting of thermophilic bacteria having optimum growth temperatures in the range of about 40 to about 45° C.

(18) The culture as described herein comprises LAB that are that are able to utilize sucrose. The thermophilic Lactobacillus acidophilus is able to utilize sucrose. Accordingly, in a preferred embodiment the frozen culture is a culture comprising Lactobacillus acidophilus, preferably a pure Lactobacillus acidophilus culture. Working example 4 herein gives an example of a stabilized pure Lactobacillus acidophilus culture.

(19) The thermophilic Streptococcus thermophilus is able to utilize sucrose. Accordingly, in a preferred embodiment the frozen culture is a mixed “Yoghurt culture” comprising Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus; or “Thermophil cheese culture” comprising Streptococcus thermophilus and Lactobacillus helveticus.

(20) Highly Concentrated Frozen Lactic Acid Bacteria Cultures

(21) The frozen cultures as described herein are, what in the food industry may be termed, highly concentrated frozen lactic acid bacteria cultures. In order to comprise sufficient bacteria such cultures should be relatively big (have a sufficient weight) combined with a relatively high concentration of viable bacteria. It is obvious that if relatively more bacteria is required the weight and/or the concentration of viable bacteria should be increased.

(22) Preferably, a frozen lactic acid bacteria (LAB) culture as described herein has a weight of at least 100 g frozen material, more preferably a weight of at least 250 g frozen material, even more preferably a weight of at least 500 g frozen material and most preferably a weight of at least 900 g frozen material. Preferably, the weight of the frozen material is less than 500 kg.

(23) Preferably, a frozen lactic acid bacteria (LAB) culture as described herein has a content of viable bacteria of at least 5×10.sup.9 colony forming units (CFU) per g frozen material, more preferably a content of viable bacteria of at least 10.sup.10 colony forming units (CFU) per g frozen material, and most preferably a content of viable bacteria of at least 2×10.sup.10 colony forming units (CFU) per g frozen material.

(24) Fermentation and suitable fermentations media for LAB are known in the art and the skilled person is capable of selecting a suitable media and fermentation conditions in relation to the specific LAB. Suitable media and fermentations are given in the working example section herein.

(25) In order to get sufficient amount of bacteria, it is in the present context preferred to make a relatively large-scale fermentation in suitable big fermentation tanks. Fermentation tanks of at least 50 l, preferably at least 90 l or bigger are preferred.

(26) After a suitable fermentation, the viable bacteria are preferably isolated by removal of the liquid (supernatant) of the fermentation media (e.g. by centrifugation). The isolated viable bacteria may be termed the isolated biomass. The isolated viable bacteria shall preferably have a content of viable bacteria of at least 10.sup.9 colony forming units (CFU) per g or ml.

(27) The frozen culture may be packaged is a suitable way in order to be provided to the user.

(28) Preferably the frozen culture is stored at a temperature from −18° C. to −60° C., more preferably from −18° C. to −50° C. The frozen culture may be stored at a temperature from −18° C. to −25° C. The freezing of the culture shall preferably be done rapidly e.g. by freezing in liquid nitrogen.

(29) Cryoprotective Agent

(30) The cryoprotective agent may preferably be selected from proteins, protein hydrolysates and amino acids. Preferred suitable examples of these include the ones selected from the group consisting of Glutamic acid, Lysine, Na-glutamate, Na-caseinate, Malt extract, Skimmed milk powder, Whey powder, Yeast extract, Gluten, Collagen, Gelatin, Elastin, Keratin, and Albumins.

(31) More preferably the cryoprotective agent is a carbonhydrate. Preferred suitable examples of these include the ones selected from the group consisting Pentoses (eg. Ribose, Xylose), Hexoses (eg. fructose, mannose, Sorbose), Disaccharides (eg. Sucrose, Trehalose, Melibiose, Lactulose), Oligosaccharides (eg. Raffinose), Oligofrutoses (eg. Actilight, Fribroloses), Polysaccharides (eg. Maltodextrins, Xanthan Gum, Pectin, Alginate, Microcrystalline cellulose, Dextran, PEG), and Sugar alcohols (Sorbitol, Manitol).

(32) The preferred carbohydrate is a disaccharide preferably Trehalose and more preferably Sucruse.

(33) A preferred mixture of cryoprotective agents is a disaccharide (preferably sucrose) plus a polysaccharide (preferably maltodextrin). Example 4 shows a thermophilic Lactobacillus acidophilus culture stabilized with a mixture of sucrose and maltodextrin. Accordingly, for a culture comprising Lactobacillus acidophilus it is preferred to use a mixture of a disaccharide (preferably sucrose) and a polysaccharide (preferably maltodextrin) as cryoprotective agents.

(34) Preferably the frozen culture comprises from 2% to 70% of a cryoprotective agent measured as w/w of the frozen material, more preferably from 3% to 50% of a cryoprotective agent measured as w/w of the frozen material, even more preferably from 4% to 40% of a cryoprotective agent measured as w/w of the frozen material and most preferably from 4% to 10% of a cryoprotective agent measured as w/w of the frozen material.

(35) The addition of the cryoprotective agent to the, after fermentation, isolated viable bacteria (biomass) may be done by mixing solid cryoprotective agent with the biomass for e.g. 30 minutes at a suitable temperature. If the cryoprotective agent is e.g. sucrose a suitable temperature may be room temperature. Alternatively a sterile solution of the cryoprotective agent may be mixed with the biomass. For sucrose suitable sterile solutions may be made from a 50% (w/w) sucrose solution. For trehalose suitable sterile solutions may be made from a 40% (w/w) solution.

(36) Use of the Frozen Lactic Acid Bacteria (LAB) Culture

(37) A frozen lactic acid bacteria (LAB) culture as described herein may be used in a process for making a food or feed product according to the art.

(38) A L-culture is preferably used to make cheese with only small holes (cottage cheese) and curdled milk products with low CO.sub.2-production.

(39) A LD-culture is preferably used to make cheese with normal hole sizes, curdled milk products (junket) and sour butter.

EXAMPLES

(40) Materials and Methods

(41) Cultures:

(42) F1 DaN, CH N 11 and CH N19 (all commercially available frozen LD-cultures, Chr. Hansen A/S, Denmark).

(43) R-603 (commercially available frozen O-culture, Chr. Hansen A/S, Denmark).

(44) La-5 (commercially available, frozen Lactobacillus acidophilus culture, Chr. Hansen A/S, Denmark).

(45) Fermentation Media and Fermentation Conditions:

(46) Medium composition for LD and O-cultures:

(47) The fermentation medium had the following composition: Casein peptone, 30 g/l; Primatone, 30 g/l; soy peptone, 30 g/l; yeast peptone, 15 g/l; MgSO.sub.4, 1.5 g/l; Na-ascorbate, 3 g/l; and lactose 50 g/l.

(48) The medium was sterilised by UHT-treatment. The finished medium had a pH of 6.5.

(49) Fermentation Condition for LD and O-Cultures:

(50) The fermentation was performed in a 100 l fermentation tank at 30° C., stirred at 50 rpm. 1% of the culture mentioned above was used as inoculum. The anaerobic fermentation was run with nitrogen in the headspace and a pressure of about 2 bar. The cultures were allowed to acidify to pH 6.2. The pH was subsequently maintained at 6.2 by controlled addition of 13.4 N NH.sub.4OH.

(51) When no further base consumption was detected, the respective culture was cooled down to about 10° C.

(52) Following cooling, each of the fermentation broths were concentrated by centrifugation and subsequently frozen as pellets in liquid nitrogen. The pellets were immediately after freezing measured for acidification activity and CFU/g and stored at −50° C. until further analysis.

(53) Media and fermentation condition for Lactobacillus acidophilus (La-5):

(54) The culture was grown in MRS broth (Merck, Damstadt, Germany) in a 100 l fermentation tank at 37° C., stirred at 20 rpm. 1% of the culture mentioned above was used as inoculum. The anaerobic fermentation was run with nitrogen in the headspace and a pressure of about 2 bar. The cultures were allowed to acidify to pH 5.5. The pH was subsequently maintained at 5.5 by controlled addition of 13.4 N NH.sub.4OH.

(55) When no further base consumption was detected, the respective culture was cooled down to about 10° C.

(56) Following cooling, each of the fermentation broths were concentrated by centrifugation and subsequently frozen as pellets in liquid nitrogen. The pellets were immediately after freezing measured for CFU/g and stored at −50° C. until further analysis.

(57) Acidifying Activity Assay and CFU Analysis:

(58) Frozen culture was inoculated on a 0.01% level in 200 ml sterilized reconstituted skimmed milk (RSM) containing 9.5% solid matter and RSM were incubated at 30° C. for 6 h to permit acidification of the substrate material. The acidification activity was measured as described by Analytical Procedure Q-AM-052, “acidification activity—UHT”, Chr. Hansen A/S (Denmark).

(59) CFU analysis was measured and calculated as described by analytical Procedure Q-AM-071, “Enumeration of microorganisms” and Q-AM-022 “Calculation of total count, Chr. Hansen A/S (Denmark) using substrate 1209—LD agar DK-med-rec-123, Chr-Hansen A/S (Denmark) or MRS agar.

Example 1: Stability Study of Frozen LD-Culture of F1 DaN and CH N11 Using Sucrose, Cystain Chloride and Sodium Citrate as Cryoprotective Agents

(60) This example describes the stability study with frozen cultures (F-DVS) of F1 DaN and CH N11 formulated with sucrose, cystein chloride and sodium citrate as cryoprotective agents. In all experiments the concentration of cystein chloride and sodium citrate were kept constant per gram concentrated biomass. The concentration of sucrose per gram biomass was varied from 6% (w/w) up to 36% (w/w). All additives were added to the concentrate as solids.

(61) After fermentation, biomass was harvested and concentrated via centrifugation from fermentation broths of CH N 11 and F1 DaN. The cell concentrate of each culture was divided into appropriate portions of 300 gram and formulated as specified in the table 1 below. The additives and concentrates were mixed for 30 minutes and subsequently freezed in liquid nitrogen and stored at −50° C. The frozen culture had a content of viable bacteria of at least 10.sup.10 colony forming units (CFU) per g frozen material. Culture activity in milk was measured the same day as formulated and followed periodically up to one year.

(62) TABLE-US-00002 TABLE 1 Formulation procedure for F-DVS of Fl DaN & CH N11. Formulation Cell Cystein Sodium Sucrose Sucrose FL DaN CH N11 ID concentrate (g) chloride (g) Citrate (g) (g) (%) (CFU/g) (CFU/g) F-DVS 300 0.00 0.00 0 0 4.0E+10 5.0E+10 F-DVS 6% 300 0.06 0.75 21 6 3.7E+10 4.7E+10 sucrose F-DVS 10% 300 0.06 0.75 36 10 3.6E+10 4.5E+10 sucrose F-DVS 22% 300 0.06 0.75 86 22 3.1E+10 3.9E+10 sucrose F-DVS 36% 300 0.06 0.75 171 36 2.5E+10 3.2E+10 sucrose

(63) Stability profiles for F-DVS of F1 DaN & CH N11 given as activity versus numbers of days and activity differences compare to day 0 are summarized in FIGS. 1 to 4. It is evident that F-DVS of F1 DaN and CH N 11 free of additives are loosing activity thus stability. The reduction in stability is equal to 0.40 pH units for CH N 11 and 0.60 pH units for F1 DaN after 365 days. All the tested sucrose formulations (6%, 10%, 22% and 36%) seem to have positive effect on the stability. Activity is reduced approx. 0.1 pH unit after 365 days positive effect on the stability. Activity is reduced approx. 0.1 pH unit after 365 days of storage at −55° C.

Example 2: Stability Study of Frozen LD-Culture of F1 DaN, CH N11 & CH N19 Using Sucrose and Trehalose as Cryoprotective Agents

(64) This example describes the stability study with frozen cultures of F1 DaN and CH N11 and CH N 19 formulated with sucrose and trehalose as cryoprotective agents. The concentration of sucrose per gram biomass was varied from 6% (w/w) up to 10% (w/w). Trehalose was only tested on a 5 (w/w) level. All sucrose concentrations were prepared from a 50% (w/w) sucrose solution added to the biomass. The trehalose concentration was prepared from a 40% (w/w) solution.

(65) After fermentation, biomass was harvested and concentrated via centrifugation from fermentation broths of F1 DaN, CH N 11 and CH N19. The cell concentrate of each culture was divided into appropriate portions of 300 gram and formulated as specified in the table 2 below. The additives and concentrates were mixed for 30 minutes and subsequently freezed in liquid nitrogen and stored at −50° C. The frozen culture had a content of viable bacteria of at least 10.sup.10 colony forming units (CFU) per g frozen material. Culture activity in milk was measured the same day as formulated and followed periodically up to 70 days.

(66) TABLE-US-00003 TABLE 2 Formulation procedure for F-DVS of Fl DaN, CH N 11 & CH N19 using sucrose and trehalose as cryoprotective agents Final Cell Additive additive Formulation concen- solution conc. Fl DaN CH N19 ID trate (g) (g) (% Sucrose) CFU/g CFU/g F-DVS 300 0 0 3.0E+10 4.0E+10 F-DVS/07G 300 43 6 2.6E+10 3.5E+10 F-DVS 5% 300 43 5 2.6E+10 3.5E+10 Trehalose F-DVS 3% 300 19 3 2.8E+10 3.8E+10 Sucrose F-DVS 5% 300 34 5 2.7E+10 3.6E+10 Sucrose F-DVS 6% 300 42 6 2.6E+10 3.5E+10 Sucrose F-DVS 8% 300 57 8 2.5E+10 3.4E+10 Sucrose F-DVS 9% 300 66 9 2.5E+10 3.3E+10 Sucrose F-DVS 10% 300 75 10 2.4E+10 3.2E+10 Sucrose F-DVS 13% 300 105 13 2.2E+10 3.0E+10 Sucrose

(67) Stability profiles for F-DVS of F1 DaN, CH N11 & CH N19 using sucrose and trehalose as cryoprotective agents are summarized in FIGS. 5 to 10.

(68) All reference cultures have lost activity (F1 DaN: 0.3 pH units after 65 days at −50° C.; CH N 11: 0.17 pH units after 60 days at −50° C.; CH N 19: 0.25 pH units after 70 days at −50° C.). All the tested formulations reduce the activity loss compare to the reference cultures. Furthermore, it is difficult to conclude which sucrose concentration is optimum with regard to stability.

(69) From the stability profiles of F1 DaN and CH N19 it can be observed that the reference and the tested sucrose formulations have an initial lost of activity within the first 1-3 weeks of storage. Hereafter, all the sucrose formulated concentrates show a constant stability profile. F1 DaN shows a higher initial loss than CH N19. However, no initial loss of activity could be observed from the stability profiles of all the tested formulations of CH N11.

Example 3: Stability Study of Frozen O-Culture of R-603

(70) Initial loss of activity within the first 1-3 weeks has so far not been seen for any of Chr. Hansen A/5 commercial available O-cultures (Lactococcus lactis subsp. cremoris & Lactococcus lactis subsp. lactis). Stability profiles for R603 followed up to 35 days and analyzed for acidification in M17 media is summarized in FIG. 11.

Example 4: Stability Study of Frozen Lactobacilus acidophislus (La-5)

(71) This example describes the stability study with frozen cultures of Lactobacillus acidophilus formulated with sucrose or sucrose and maltodextrine as cryoprotective agents. The concentration of sucrose per gram biomass was 32% (w/w). Sucrose and maltodextrine were tested at 16%/16% (w/w) level.

(72) After fermentation, biomass was harvested and concentrated by centrifugation. The cell concentrate was divided into appropriate portions of 300 gram and formulated. The additives and concentrates were mixed for 30 minutes and subsequently freezed in liquid nitrogen and stored at −20, −50 or −80° C. The stability was measured as colony-forming units (CFU) per g frozen material on MRS agar 37° C. (72 hours).

(73) TABLE-US-00004 TABLE 3 Lactobacillus acidophilus stored at different temperatures. Storage stability is measured as CFU/g after 6, 13, and 68 days Temp = −20° Temp = −50° Temp = −80° +32% Sucrose Days C. C. C. at −20° C. 0 4.35E+10 4.35E+10 4.35E+10 3.82E+10 6 3.14E+10 4.87E+10 4.77E+10 2.61E+10 13 2.75E+10 4.37E+10 4.61E+10 2.50E+10 68 6.52E+09 4.15E+10 4.14E+10 2.51E+10

(74) TABLE-US-00005 TABLE 4 Lactobacillus acidophilus stored at −20° C. without additives and with 32% sucrose or with 16% sucrose + 16% maltodextrine. without 32% 16% sucrose + Days additives sucrose 16% maltodextrine 0 2.81E+10 3.02E+10 3.82E+10 7 1.81E+10 1.99E+10 2.97E+10 14 1.11E+10 1.56E+10 2.04E+10 21 out of range 1.98E+10 2.93E+10 54 8.60E+09 1.53E+10 2.54E+10

(75) Lactobacillus acidophilus seems storage stable at −50 and −80° C., but the viability is declining if the culture is stored at −20° C. By use of additives; sucrose or sucrose and maltodextrine—it is possible to improve the stability of the culture at −20° C.

REFERENCES

(76) EP 259739 A1, Miles Laboratories, 16 Mar. 1988 F. J. Chavarri et al, “Cryoprotective agents for frozen concentrated starters from non-bitter Streptococcus Lactis strains”, Biotechnology letters, vol 10, 1, 11-16 (1988) R. Cárcoba et al., “Influence of cryoprotectants on the viability and acidifying activity of frozen and freeze-dried cells of the novel starter strain Lactococcus lactis subsp. lactis CECT 5180”, Eur Food Res Technol (2000) 211, 433-437 WO 00/39281, Chr. Hansen A/S, 6 Jul. 2000