US20060204484A1 - Storage stable frozen lactic acid bacteria culture - Google Patents
Storage stable frozen lactic acid bacteria culture Download PDFInfo
- Publication number
- US20060204484A1 US20060204484A1 US10/543,240 US54324004A US2006204484A1 US 20060204484 A1 US20060204484 A1 US 20060204484A1 US 54324004 A US54324004 A US 54324004A US 2006204484 A1 US2006204484 A1 US 2006204484A1
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- United States
- Prior art keywords
- culture
- frozen
- lab
- sucrose
- lactic acid
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Abandoned
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2400/00—Lactic or propionic acid bacteria
- A23V2400/11—Lactobacillus
- A23V2400/113—Acidophilus
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2400/00—Lactic or propionic acid bacteria
- A23V2400/21—Streptococcus, lactococcus
- A23V2400/215—Cremoris
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2400/00—Lactic or propionic acid bacteria
- A23V2400/21—Streptococcus, lactococcus
- A23V2400/219—Diacetilactis
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2400/00—Lactic or propionic acid bacteria
- A23V2400/21—Streptococcus, lactococcus
- A23V2400/231—Lactis
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2400/00—Lactic or propionic acid bacteria
- A23V2400/31—Leuconostoc
- A23V2400/317—Cremoris
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2400/00—Lactic or propionic acid bacteria
- A23V2400/31—Leuconostoc
- A23V2400/321—Mesenteroides
Definitions
- the present invention relates to 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 9 colony forming units (CFU) per g frozen material.
- LAB storage stable frozen lactic acid bacteria
- Microorganisms are involved in the manufacture of food and feed products including most dairy products.
- bacterial cultures in particular cultures of bacteria that are generally classified as lactic acid bacteria are essential in the making of all fermented milk products, cheese and butter. Cultures of such bacteria may be referred to as starter cultures and they impart specific features to various dairy products by performing a number of functions.
- lactic acid bacteria designates a group of Gram positive, catalase negative, non-motile, microaerophilic or anaerobic bacteria which ferment sugar with the production of acids including lactic acid as the predominantly produced acid, acetic acid, formic acid and propionic acid.
- the industrially most useful lactic acid bacteria are found among Lactococcus species, Streptococcus species, Enterococcus species, Lactobacillus species, Leuconostoc species and Pediococcus species.
- dairy starter culture strains of lactic acid bacteria are generally divided into mesophilic organisms having optimum growth temperatures at about 30° C. and thermophilic organisms having optimum growth temperatures in the range of about 40 to about 45° C.
- Typical organisms belonging to the mesophilic group include Lactococcus lactis subsp. lactis, Lactococcus lactis subsp. cremoris, Leuconostoc mesenteroides subsp. cremoris, Pediococcus pentosaceus, Lactococcus lactis subsp. lactis biovar. diacetylactis and Lactobacillus casei subsp. casei .
- Thermophilic lactic acid bacterial species include as examples Streptococcus thermophilus, Enterococcus faecium, Lactobacillus lactis, Lactobacillus helveticus, Lactobacillus delbrueckii subsp. bulgaricus and Lactobacillus acidophilus.
- the dairy starter cultures are also classified according to their specific species composition and preferred industrial use.
- a pure starter culture comprises only a single specie and a mixed culture comprises two or more different species.
- Commercial relevant mesophilic mixed cultures include:
- An O-culture is used to make cheese without holes (Cheddar, Cheshire, Feta).
- a D-culture is used to make butter.
- a L-culture is used to cheese with only small holes (cottage cheese) and curdled milk products with low CO 2 -production.
- a LD-culture is used to make cheese with normal hole sizes, curdled milk products (junket) and sour butter. Commercially, a LD-culture is currently one of the most used mixed cultures.
- thermophilic mixed cultures include:
- Yoghurt culture is used to make yoghurt and special Italian cheeses.
- An Thermophil cheese culture is used to make emmentaler cheese and special Italian cheeses.
- a “bulk starter” is defined herein as a starter culture propagated at the dairy plant for inoculation into milk. Highly concentrated cultures may be referred to as direct vat set (DVS)-cultures.
- a commercial relevant highly concentrated frozen culture generally has a weight of at least 50 g and a content of viable bacteria of at least 10 9 colony forming units (CFU) per g.
- DVS-starter cultures Another presentation of commercial highly concentrated DVS-starter cultures is as freeze-dried or lyophilized cultures in the form of a powder. In this form, the starter can be shipped without refrigeration.
- the present inventors are not aware of any commercial available highly concentrated frozen cultures that comprise significant amounts of cryoprotective agents.
- EP259739 describes different suitable cryoprotective agents for freeze-dried cultures.
- a freeze-dried culture in the form of a powder is physically significant different from a frozen culture among others due to that a freeze-dried powder comprises significant less water as compared to a frozen culture. Accordingly, it is submitted that the skilled person would prima facie not consider that a specific cryoprotective agent described as useful for a freeze-dried culture would also be similar useful in a frozen culture.
- WO00/39281 (Chr. Hansen A/S) describes a liquid starter culture stabilized by different cryoprotective agents. Page 5, lines 5-7 reads “the expression “liquid starter culture” relates to non-frozen liquid starter cultures having a liquid phase, e.g. an aqueous phase, content that is typically in the range of 50-90% by weight”. Consequently, the liquid culture described in WO00/39281 is a non-frozen culture.
- the present inventors Prior to the present invention, the present inventors believed that there were no significant storage stability problems in relation to commercially relevant highly concentrated frozen lactic acid bacteria cultures. As said above, the present inventors are not aware of any commercial available highly concentrated frozen cultures that comprise significant amounts of cryoprotective agents.
- an O-culture comprises Lactococcus lactis subsp. lactis and Lactococcus lactis subsp. cremoris and a LD-culture comprises Lactococcus lactis subsp. lactis, Lactococcus lactis subsp. cremoris, Lactococcus lactis subsp. lactis biovar. diacetylactis and Leuconostoc mesenteroides subsp. cremoris.
- the Leuconostoc mesenteroides subsp. cremoris present in the LD-culture is able to utilize sucrose.
- the O-culture does not comprise bacteria that are able to utilize sucrose.
- a commercial relevant highly concentrated frozen culture generally has a weight of at least 50 g and a content of viable bacteria of at least 10 9 colony forming units (CFU) per g.
- CFU colony forming units
- a first aspect of the invention relates to a 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 9 colony forming units (CFU) per g frozen material, characterized in that the frozen culture comprises from 0.5% to 80% of a cryoprotective agent measured as w/w of the frozen material.
- LAB frozen lactic acid bacteria
- the cryoprotective agent should preferably be added to the viable bacteria before they are frozen.
- the invention relates to a method for making a 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 109 colony forming units (CFU) per g frozen material comprising following steps:
- a third aspect of the invention relates to a frozen lactic acid bacteria (LAB) culture obtainable by the method for making a frozen lactic acid bacteria (LAB) culture of the second aspects of the invention.
- LAB frozen lactic acid bacteria
- a fourth aspect of the invention relates to use of the frozen lactic acid bacteria (LAB) culture as described above in a process for making a food or feed product.
- LAB frozen lactic acid bacteria
- LAB that are that are able to utilize sucrose denotes LAB that are able to ferment the sugar sucrose with the production of acids.
- the term “material” of the culture denotes the relevant substances of the culture including both the viable bacteria and cryoprotective agent. Possible packing is not included. Consequently, the weight of the material of the culture is not including the weight of possible packing.
- packing should be understood broadly. It denotes that the frozen lactic acid bacteria (LAB) culture should be packed in order could to be provided to the user. It may be packed in a bottle, a tetra-pack, etc.
- LAB frozen lactic acid bacteria
- cryoprotective agent denotes a substance that is able to improve the storage stability of the frozen culture. In the present context it may be a single specific cryoprotective agents or it may be two or more different agents. Accordingly, the w/w percentage of the cryoprotective agent(s) within the culture material should be understood as the sum of the amount of cryoprotective agent(s).
- a preferred way to determine whether a substance is a cryoprotective agent that is able to improve the storage stability of the frozen culture is to spilt a culture, as described herein, in two samples, add a specified amount of the cryoprotective agent to one of them, freezing both of them and measure the milk acidifying activity of the cultures on the same day as freezing and periodically up to one year under frozen storage. If the culture with cryoprotective agent has improved milk acidifying activity seen over the storage period the substance is a cryoprotective agent.
- a suitable milk acidifying activity assay is given in working examples herein.
- FIG. 1 to 4 Stability profiles for F-DVS of Fl DaN & CH N11. For further details see working Example 1.
- FIG. 5 to 10 Stability profiles for F-DVS of Fl DaN, CH N11 & CH N19. For further details see working Example 2.
- FIG. 11 Stability profiles for R603. For further details see working Example 3.
- LAB culture denotes a mixed culture that comprises two or more different LAB species.
- a “pure lactic acid bacteria (LAB) culture” denotes a pure culture that comprises only a single LAB species specie.
- the culture as described herein may be a mesophilic culture consisting of mesophilic bacteria having optimum growth temperatures at about 30° C.
- 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.
- 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.
- a L-culture comprises Lactococcus lactis subsp. lactis, Lactococcus lactis subsp. cremoris and Leuconostoc mesenteroides subsp. cremoris.
- a LD-culture comprises Lactococcus lactis subsp. lactis, Lactococcus lactis subsp. cremoris, Lactococcus lactis subsp. lactis biovar. diacetylactis and Leuconostoc mesenteroides subsp. cremoris.
- 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.
- Lactococcus lactis subsp. lactis biovar. diacetylactis and Leuconostoc mesenteroides subsp. cremoris could be preferred.
- a preferred LD-culture comprises: Lactococcus lactis subsp. lactis , 60-95%, preferably Lactococcus lactis subsp. cremoris 70-90% Lactococcus lactis subsp. lactis biovar. 5-40%, preferably diacetylactis , Leuconostoc mesenteroides 10 to 30% subsp. cremoris
- 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.
- the culture as described herein comprises LAB that are that are able to utilize sucrose.
- the thermophilic Lactobacillus acidophilus is able to utilize sucrose.
- 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.
- thermophilic Streptococcus thermophilus is able to utilize sucrose. Accordingly, in a preferred embodiment the frozen culture is a mixed
- 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.
- 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.
- the weight of the frozen material is less than 500 kg.
- a frozen lactic acid bacteria (LAB) culture as described herein has a content of viable bacteria of at least 5 ⁇ 10 9 colony forming units (CFU) per g frozen material, more preferably a content of viable bacteria of at least 1010 colony forming units (CFU) per g frozen material, and most preferably a content of viable bacteria of at least 2 ⁇ 10 10 colony forming units (CFU) per g frozen material.
- CFU colony forming units
- 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.
- Fermentation tanks of at least 50 l, preferably at least 90 l or bigger are preferred.
- 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 9 colony forming units (CFU) per g or ml.
- the frozen culture may be packaged is a suitable way in order to be provided to the user.
- 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.
- 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.
- 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).
- Pentoses eg. Ribose, Xylose
- Hexoses eg. fructose, mannose, Sorbose
- Disaccharides eg. Sucrose, Trehalose, Meli
- the preferred carbohydrate is a disaccharide preferably Trehalose and more preferably Sucrose.
- 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.
- 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.
- cryoprotective agent added 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.
- a suitable temperature may be room temperature.
- a sterile solution of the cryoprotective agent may be mixed with the biomass.
- sucrose suitable sterile solutions may be made from a 50% (w/w) sucrose solution.
- trehalose suitable sterile solutions may be made from a 40% (w/w) solution.
- 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.
- a L-culture is preferably used to make cheese with only small holes (cottage cheese) and curdled milk products with low CO 2 -production.
- a LD-culture is preferably used to make cheese with normal hole sizes, curdled milk products (junket) and sour butter.
- R-603 (commercially available frozen O-culture, Chr. Hansen A/S, Denmark).
- La-5 commercially available frozen Lactobacillus acidophilus culture, Chr. Hansen A/S, Denmark.
- the fermentation medium had the following composition: Casein peptone, 30 g/l; Primatone, 30 g/l; soy peptone, 30 g/l; yeast peptone, 15 ⁇ l; MgSO 4 , 1.5 g/l; Na-ascorbate, 3 g/l; and lactose 50 g/l.
- the medium was sterilised by UHT-treatment.
- the finished medium had a pH of 6.5.
- 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 NH 4 OH.
- the respective culture was cooled down to about 10° C.
- 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.
- 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 4 OH.
- the respective culture was cooled down to about 10° C.
- 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.
- 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).
- 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.
- This example describes the stability study with frozen cultures (F-DVS) of Fl DaN and CH N11 formulated with sucrose, cystein chloride and sodium citrate as cryoprotective agents.
- F-DVS frozen cultures
- concentration of cystein chloride and sodium citrate were kept constant per gram concentrated biomass.
- 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.
- Stability profiles for F-DVS of Fl DaN & CH N 11 given as activity versus numbers of days and activity differences compare to day 0 are summarized in FIG. 1 to 4 . It is evident that F-DVS of Fl 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 Fl 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 of storage at ⁇ 55° C.
- This example describes the stability study with frozen cultures of Fl 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.
- This example describes the stability study with frozen cultures of Lactobacillus acidophilus formulated with sucrose or sucrose and maltodextrine as cryoprotective agents.
- concentration of sucrose per gram biomass was 32% (w/w).
- Sucrose and maltodextrine were tested at 16%/16% (w/w) level.
- CFU colony-forming units
- Lactobacillus acidophilus seems storage stable at ⁇ 50 and ⁇ 80° C., but the viability is declining if the culture is stored at ⁇ 20° C.
- sucrose or sucrose and maltodextrine it is possible to improve the stability of the culture at ⁇ 20° C.
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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 109 colony forming units (CFU) per g frozen material.
Description
- The present invention relates to 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 109 colony forming units (CFU) per g frozen material.
- Microorganisms are involved in the manufacture of food and feed products including most dairy products. Thus, bacterial cultures, in particular cultures of bacteria that are generally classified as lactic acid bacteria are essential in the making of all fermented milk products, cheese and butter. Cultures of such bacteria may be referred to as starter cultures and they impart specific features to various dairy products by performing a number of functions.
- Commercial dairy starter cultures are generally composed of lactic acid and citric acid fermenting lactic acid bacteria. In the present context, the expression “lactic acid bacteria” designates a group of Gram positive, catalase negative, non-motile, microaerophilic or anaerobic bacteria which ferment sugar with the production of acids including lactic acid as the predominantly produced acid, acetic acid, formic acid and propionic acid. The industrially most useful lactic acid bacteria are found among Lactococcus species, Streptococcus species, Enterococcus species, Lactobacillus species, Leuconostoc species and Pediococcus species.
- Commonly used dairy starter culture strains of lactic acid bacteria are generally divided into mesophilic organisms having optimum growth temperatures at about 30° C. and thermophilic organisms having optimum growth temperatures in the range of about 40 to about 45° C.
- Typical organisms belonging to the mesophilic group include Lactococcus lactis subsp. lactis, Lactococcus lactis subsp. cremoris, Leuconostoc mesenteroides subsp. cremoris, Pediococcus pentosaceus, Lactococcus lactis subsp. lactis biovar. diacetylactis and Lactobacillus casei subsp. casei. Thermophilic lactic acid bacterial species include as examples Streptococcus thermophilus, Enterococcus faecium, Lactobacillus lactis, Lactobacillus helveticus, Lactobacillus delbrueckii subsp. bulgaricus and Lactobacillus acidophilus.
- The dairy starter cultures are also classified according to their specific species composition and preferred industrial use. A pure starter culture comprises only a single specie and a mixed culture comprises two or more different species. Commercial relevant mesophilic mixed cultures include:
-
- “O-culture” comprising Lactococcus lactis subsp. lactis and Lactococcus lactis subsp. cremoris.
- “D-culture” comprising Lactococcus lactis subsp. lactis, Lactococcus lactis subsp. cremoris and Lactococcus lactis subsp. lactis biovar. diacetylactis.
- “L-culture” comprising Lactococcus lactis subsp. lactis, Lactococcus lactis subsp. cremoris and Leuconostoc mesenteroides subsp. cremoris.
- “LD-culture” comprising Lactococcus lactis subsp. lactis, Lactococcus lactis subsp. cremoris, Lactococcus lactis subsp. lactis biovar. diacetylactis and Leuconostoc mesenteroides subsp. cremoris.
- An O-culture is used to make cheese without holes (Cheddar, Cheshire, Feta). A D-culture is used to make butter. A L-culture is used to cheese with only small holes (cottage cheese) and curdled milk products with low CO2-production. A LD-culture is used to make cheese with normal hole sizes, curdled milk products (junket) and sour butter. Commercially, a LD-culture is currently one of the most used mixed cultures.
- Commercial relevant thermophilic mixed cultures include:
-
- “Yoghurt culture” comprising Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus.
- “Thermophil cheese culture” comprising Streptococcus thermophilus and Lactobacillus helveticus.
- An Yoghurt culture is used to make yoghurt and special Italian cheeses. An Thermophil cheese culture is used to make emmentaler cheese and special Italian cheeses.
- Commercial starter cultures may commonly be distributed as frozen cultures. Highly concentrated frozen cultures are commercially very interesting since such cultures can be inoculated directly into milk without intermediate transfer. In others words, such highly concentrated frozen cultures comprises so many bacteria that dairies do not have to make in-house bulk starters. A “bulk starter” is defined herein as a starter culture propagated at the dairy plant for inoculation into milk. Highly concentrated cultures may be referred to as direct vat set (DVS)-cultures.
- In order to comprise sufficient bacteria a commercial relevant highly concentrated frozen culture generally has a weight of at least 50 g and a content of viable bacteria of at least 109 colony forming units (CFU) per g.
- Another presentation of commercial highly concentrated DVS-starter cultures is as freeze-dried or lyophilized cultures in the form of a powder. In this form, the starter can be shipped without refrigeration.
- The article of F. J. Chavarri et al (Biotechnology letters,
vol 10, 1, 11-16 (1988), “Cryoprotective agents for frozen concentrated starters from non-bitter Streptococcus lactis strains”) describes that the storage viability of a frozen pure Streptococcus lactis culture could be improved by addition of 5% lactose or 5% sucrose. The lactose or sucrose worked as cryoprotective agents. Streptococcus lactis is a former name of Lactococcus lactis subsp. lactis. - Similarly, the article of R. Cárcoba et al (Eur Food Res Technol (2000) 211, 433-437, “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”) describes that the storage viability of a frozen pure Lactococcus lactis subsp. lactis culture could be improved by addition of different cryoprotective agents such as sugars (lactose, sucrose and trehalose), glutamic acid and gelatin.
- The present inventors are not aware of any commercial available highly concentrated frozen cultures that comprise significant amounts of cryoprotective agents.
- EP259739 describes different suitable cryoprotective agents for freeze-dried cultures. A freeze-dried culture in the form of a powder is physically significant different from a frozen culture among others due to that a freeze-dried powder comprises significant less water as compared to a frozen culture. Accordingly, it is submitted that the skilled person would prima facie not consider that a specific cryoprotective agent described as useful for a freeze-dried culture would also be similar useful in a frozen culture.
- WO00/39281 (Chr. Hansen A/S) describes a liquid starter culture stabilized by different cryoprotective agents.
Page 5, lines 5-7 reads “the expression “liquid starter culture” relates to non-frozen liquid starter cultures having a liquid phase, e.g. an aqueous phase, content that is typically in the range of 50-90% by weight”. Consequently, the liquid culture described in WO00/39281 is a non-frozen culture. - Prior to the present invention, the present inventors believed that there were no significant storage stability problems in relation to commercially relevant highly concentrated frozen lactic acid bacteria cultures. As said above, the present inventors are not aware of any commercial available highly concentrated frozen cultures that comprise significant amounts of cryoprotective agents.
- Prior to the present invention, stability studies had been made starting from commercial highly concentrated lactic acid bacteria cultures that already had been frozen for 2-3 month. For instance, studies were made on 2-3 month old frozen LD-cultures. These studies showed no significant degradation of activity of the LD-cultures over a period of one year at temperature below −45° C. Consequently, it was believed that commercial relevant LD-cultures did not have significant storage stability problems.
- In order to analyze the stability during the first weeks of frozen storage different O-cultures were analyzed right from the first day of frozen storage. They did not show any significant reduction of activity neither during the first weeks nor during the next 12 month. Accordingly, these data essentially confirmed that there should be no storage stability problems in relation to commercially relevant highly concentrated frozen cultures.
- Despite this, the present inventors continued to investigate stability issues of different commercial relevant frozen cultures and after a number of studies they identified that for instance frozen LD-cultures had a significant loss of activity within the first 1-3 weeks of frozen storage. After these weeks the further loss of activity was relatively insignificant in line with the prior known results described above.
- In summary, the work of the present inventors has identified hitherto unrecognized storage stability problems in relation to some types of commercial relevant highly concentrated frozen lactic acid bacteria cultures, such as e.g. commercial available frozen LD-cultures.
- Once having identified this problem, the present inventors could start to try to solve the problem. As described herein they solved this by addition of cryoprotective agents to the relevant highly concentrated frozen cultures.
- As said above an O-culture comprises Lactococcus lactis subsp. lactis and Lactococcus lactis subsp. cremoris and a LD-culture comprises Lactococcus lactis subsp. lactis, Lactococcus lactis subsp. cremoris, Lactococcus lactis subsp. lactis biovar. diacetylactis and Leuconostoc mesenteroides subsp. cremoris.
- The Leuconostoc mesenteroides subsp. cremoris present in the LD-culture is able to utilize sucrose. The O-culture does not comprise bacteria that are able to utilize sucrose.
- Consequently, without being limited to theory, it is believed that the herein identified stability problems relate to commercial relevant highly frozen cultures that comprise bacteria that are able to utilize sucrose.
- As said above, in order to comprise sufficient bacteria a commercial relevant highly concentrated frozen culture generally has a weight of at least 50 g and a content of viable bacteria of at least 109 colony forming units (CFU) per g. The pure Lactococcus lactis subsp. lactis cultures described in the articles of F. J. Chavarri et al and R. Carboba et al (see above) are in the present context not considered commercial relevant highly concentrated frozen cultures since they are made on must smaller scale and comprises significant less grams of frozen culture.
- Accordingly, a first aspect of the invention relates to a 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 109 colony forming units (CFU) per g frozen material, characterized in that the frozen culture comprises from 0.5% to 80% of a cryoprotective agent measured as w/w of the frozen material.
- The cryoprotective agent should preferably be added to the viable bacteria before they are frozen.
- Accordingly, in a second aspect the invention relates to a method for making a 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 109 colony forming units (CFU) per g frozen material comprising following steps:
-
- (i) adding a cryoprotective agent to viable bacteria to get at least 50 g of material with a content of viable bacteria of at least 109 colony forming units (CFU) per g material and comprising the cryoprotective agent in an amount from 0.5% to 80% measured as w/w of the material,
- (ii) freezing the material to get frozen material, and
- (iii) packing the frozen material in a suitable way.
- A third aspect of the invention relates to a frozen lactic acid bacteria (LAB) culture obtainable by the method for making a frozen lactic acid bacteria (LAB) culture of the second aspects of the invention.
- A fourth aspect of the invention relates to use of the frozen lactic acid bacteria (LAB) culture as described above in a process for making a food or feed product.
- Definitions:
- Prior to a discussion of the detailed embodiments of the invention is provided a definition of specific terms related to the main aspects of the invention.
- The term “LAB that are that are able to utilize sucrose” denotes LAB that are able to ferment the sugar sucrose with the production of acids.
- The term “material” of the culture denotes the relevant substances of the culture including both the viable bacteria and cryoprotective agent. Possible packing is not included. Consequently, the weight of the material of the culture is not including the weight of possible packing.
- The term “packing” should be understood broadly. It denotes that the frozen lactic acid bacteria (LAB) culture should be packed in order could to be provided to the user. It may be packed in a bottle, a tetra-pack, etc.
- The term “a cryoprotective agent” denotes a substance that is able to improve the storage stability of the frozen culture. In the present context it may be a single specific cryoprotective agents or it may be two or more different agents. Accordingly, the w/w percentage of the cryoprotective agent(s) within the culture material should be understood as the sum of the amount of cryoprotective agent(s). A preferred way to determine whether a substance is a cryoprotective agent that is able to improve the storage stability of the frozen culture is to spilt a culture, as described herein, in two samples, add a specified amount of the cryoprotective agent to one of them, freezing both of them and measure the milk acidifying activity of the cultures on the same day as freezing and periodically up to one year under frozen storage. If the culture with cryoprotective agent has improved milk acidifying activity seen over the storage period the substance is a cryoprotective agent. A suitable milk acidifying activity assay is given in working examples herein.
- Embodiments of the present invention is described below, by way of examples only
-
FIG. 1 to 4: Stability profiles for F-DVS of Fl DaN & CH N11. For further details see working Example 1. -
FIG. 5 to 10: Stability profiles for F-DVS of Fl DaN, CH N11 & CH N19. For further details see working Example 2. -
FIG. 11 : Stability profiles for R603. For further details see working Example 3. - A frozen Lactic Acid Bacteria (LAB) Culture
- 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.
- The culture as described herein may be a mesophilic culture consisting of mesophilic bacteria having optimum growth temperatures at about 30° C.
- 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.
- 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.
- A L-culture comprises Lactococcus lactis subsp. lactis, Lactococcus lactis subsp. cremoris and Leuconostoc mesenteroides subsp. cremoris.
- A LD-culture comprises Lactococcus lactis subsp. lactis, Lactococcus lactis subsp. cremoris, Lactococcus lactis subsp. lactis biovar. diacetylactis and Leuconostoc mesenteroides subsp. cremoris.
- 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.
- 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.
- A preferred LD-culture comprises:
Lactococcus lactis subsp. lactis, 60-95%, preferably Lactococcus lactis subsp. cremoris 70-90% Lactococcus lactis subsp. lactis biovar. 5-40%, preferably diacetylactis, Leuconostoc mesenteroides 10 to 30% subsp. cremoris - 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.
- 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.
- 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.
- 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.
- 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.
Highly Concentrated Frozen Lactic Acid Bacteria Cultures
- 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.
- 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.
- Preferably, a frozen lactic acid bacteria (LAB) culture as described herein has a content of viable bacteria of at least 5×109 colony forming units (CFU) per g frozen material, more preferably a content of viable bacteria of at least 1010 colony forming units (CFU) per g frozen material, and most preferably a content of viable bacteria of at least 2×1010 colony forming units (CFU) per g frozen material.
- 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.
- 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.
- 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 109 colony forming units (CFU) per g or ml.
- The frozen culture may be packaged is a suitable way in order to be provided to the user.
- 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.
- Cryoprotective Agent
- 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.
- 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).
- The preferred carbohydrate is a disaccharide preferably Trehalose and more preferably Sucrose.
- 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.
- 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.
- 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.
- Use of the Frozen Lactic Acid Bacteria (LAB) Culture
- 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.
- A L-culture is preferably used to make cheese with only small holes (cottage cheese) and curdled milk products with low CO2-production.
- A LD-culture is preferably used to make cheese with normal hole sizes, curdled milk products (junket) and sour butter.
- Materials and Methods
- Cultures:
- Fl DaN,
CH N 11 and CH N19 (all commercially available frozen LD-cultures, Chr. Hansen A/S, Denmark). - R-603 (commercially available frozen O-culture, Chr. Hansen A/S, Denmark).
- La-5 (commercially available frozen Lactobacillus acidophilus culture, Chr. Hansen A/S, Denmark).
- Fermentation Media and Fermentation Conditions:
- Medium composition for LD and O-cultures:
- The fermentation medium had the following composition: Casein peptone, 30 g/l; Primatone, 30 g/l; soy peptone, 30 g/l; yeast peptone, 15 μl; MgSO4, 1.5 g/l; Na-ascorbate, 3 g/l; and lactose 50 g/l.
- The medium was sterilised by UHT-treatment. The finished medium had a pH of 6.5.
- Fermentation Condition for LD and O-Cultures:
- 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 NH4OH.
- When no further base consumption was detected, the respective culture was cooled down to about 10° C.
- 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.
- Media and Fermentation Condition for Lactobacillus acidophilus (La-5):
- 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 NH4OH.
- When no further base consumption was detected, the respective culture was cooled down to about 10° C.
- 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.
- Acidifying Activity Assay and CFU Analysis:
- 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).
- 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.
- This example describes the stability study with frozen cultures (F-DVS) of Fl 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.
- After fermentation, biomass was harvested and concentrated via centrifugation from fermentation broths of
CH N 11 and Fl 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 1010 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.TABLE 1 Formulation procedure for F-DVS of Fl DaN & CH N11. Cell Cystein Sodium Formulation concentrate chloride Citrate Sucrose Sucrose FL DaN CH N11 ID (g) (g) (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 - Stability profiles for F-DVS of Fl DaN &
CH N 11 given as activity versus numbers of days and activity differences compare today 0 are summarized inFIG. 1 to 4. It is evident that F-DVS of Fl DaN andCH N 11 free of additives are loosing activity thus stability. The reduction in stability is equal to 0.40 pH units forCH N 11 and 0.60 pH units for Fl 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 of storage at −55° C. - This example describes the stability study with frozen cultures of Fl 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.
- After fermentation, biomass was harvested and concentrated via centrifugation from fermentation broths of Fl 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 1010 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.TABLE 2 Formulation procedure for F-DVS of Fl DaN, CH N 11 & CHN19 using sucrose and trehalose as cryoprotective agents Final Cell Additive additive Formulation concentrate solution conc. Fl DaN CH N19 ID (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 - Stability profiles for F-DVS of Fl DaN, CH N11 & CH N 19 using sucrose and trehalose as cryoprotective agents are summarized in
FIG. 5 to 10. - All reference cultures have lost activity (Fl 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.
- From the stability profiles of Fl 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. Fl 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.
- Initial loss of activity within the first 1-3 weeks has so far not been seen for any of Chr. Hansen A/S 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 . - 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.
- 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).
TABLE 3 Lactobacillus acidophilus stored at different temperatures. Storage stability is measured as CFU/g after 6, 13, and 68 days +32% Sucrose Temp = −20° Temp = −50° Temp = −80° at −20° Days C. C. C. 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 -
TABLE 4 Lactobacillus acidophilus stored at −20° C. without additives and with 32% sucrose or with 16% sucrose + 16% maltodextrine. without a 16% sucrose + 16% Days tives 32 % sucrose 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 - 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.
-
- 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
Claims (20)
1. A frozen lactic acid bacteria (LAB) culture that comprises LAB that are able to utilize sucrose and is a mixed mesophilic culture consisting of mesophilic bacteria having optimum growth temperatures at about 30° C., has a weight of at least 50 g frozen material and a content of viable bacteria of at least 109 colony forming units (CFU) per g frozen material and characterized in that the frozen culture comprises from 0.5% to 80% of a cryoprotective agent measured as w/w of the frozen material.
2. The frozen culture of claim 1 , wherein the culture is a LD-culture that comprises 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 a culture comprising thermophilic Lactobacillus acidophilus.
4. The frozen culture of claim 1 , wherein the cryoprotective agent is a carbohydrate, preferably a disaccharide.
5. The frozen culture of claim 4 , wherein the disaccharide is trehalose or sucrose.
6. The frozen culture of claim 4 , wherein the cryoprotective agent is a mixture of a disaccharide, preferably sucrose, and a polysaccharide, preferably maltodextrine.
7. A method for making a frozen lactic acid bacteria (LAB) culture that comprises LAB 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 109 colony forming units (CFU) per g frozen material comprising following steps:
(i) adding a cryoprotective agent to viable bacteria to get at least 50 g of material with a content of viable bacteria of at least 109 colony forming units (CFU) per g material and comprising the cryoprotective agent in an amount from 0.5% to 80% measured as w/w of the material,
(ii) freezing the material to get frozen material, and
(iii) packing the frozen material in a suitable way.
8. A frozen lactic acid bacteria (LAB) culture obtainable by the method for making a frozen lactic acid bacteria (LAB) culture of claim 7 .
9. Use of the frozen lactic acid bacteria (LAB) culture of claim 1 9 in a process for making a food or feed product.
10. A commercially viable, highly concentrated lactic acid bacteria (LAB) culture adapted to be frozen, the culture comprising:
lactic acid bacteria (LAB) of at least 1×109 colony forming units (CFU) per gram of culture; and,
a cryoprotective agent; wherein the cryoprotective agent is 0.5% to 80% (w/w) of the culture;
wherein the culture has a weight of at least 50 grams; and,
wherein the LAB are adapted to utilize sucrose; and are mesophilic having optimum growth temperatures of about 30° C.
11. A culture according to claim 10 , wherein the LAB are one of pure or mixed.
12. A culture according to claim 10 , wherein the LAB includes a Leuconostoc mesenteroides subsp. cremoris.
13. A culture according to claim 10 , wherein the culture is one of an L-culture and an LD-culture.
14. A culture according to claim 10 , wherein the LAB are mesophilic having optimum growth temperatures of about 30° C.
15. A culture according to claim 10 , wherein the LAB includes a thermophilic Lactobacillus acidophilus.
16. A culture according to claim 10 , wherein the LAB includes a thermophilic Streptococcus thermophilus.
17. A culture according to claim 10 , wherein the cryoprotective agent is one or more of a carbohydrate, a disaccharide, a polysaccharide, trehalose, sucrose or maltodextrin.
18. A culture according to claim 10 , wherein the cryoprotective agent is a mixture of one of a disaccharide and sucrose, with one of a polysaccharide and maltodextrin.
19. A culture according to claim 10 , wherein the cryoprotective agent includes one of a sterile 50% (w/w) sucrose solution and a sterile 40% (w/w) trehalose solution.
20. A culture according to claim 10 , wherein the cryoprotective agent is selected from proteins, protein hydrolysates and amino acids.
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US14/587,953 US9848615B2 (en) | 2003-01-22 | 2014-12-31 | Storage stable frozen lactic acid bacteria culture |
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PCT/DK2004/000025 WO2004065584A1 (en) | 2003-01-22 | 2004-01-19 | Storage stable frozen lactic acid bacteria culture |
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US20060204484A1 true US20060204484A1 (en) | 2006-09-14 |
Family
ID=32524184
Family Applications (2)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US10/543,240 Abandoned US20060204484A1 (en) | 2003-01-22 | 2004-01-19 | Storage stable frozen lactic acid bacteria culture |
US14/587,953 Expired - Lifetime US9848615B2 (en) | 2003-01-22 | 2014-12-31 | Storage stable frozen lactic acid bacteria culture |
Family Applications After (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US14/587,953 Expired - Lifetime US9848615B2 (en) | 2003-01-22 | 2014-12-31 | Storage stable frozen lactic acid bacteria culture |
Country Status (5)
Country | Link |
---|---|
US (2) | US20060204484A1 (en) |
EP (2) | EP1441027A1 (en) |
DK (1) | DK1587909T3 (en) |
ES (1) | ES2427542T3 (en) |
WO (1) | WO2004065584A1 (en) |
Cited By (10)
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US20110020494A1 (en) * | 2007-10-30 | 2011-01-27 | Lars Petersen | Growth medium for lactic acid bacteria |
US20120003721A1 (en) * | 2009-02-23 | 2012-01-05 | Birgitte Yde | Method for making lactic acid bacteria composition |
EP2649175B1 (en) | 2010-12-10 | 2017-11-29 | DSM IP Assets B.V. | Starter culture compositions |
US9848615B2 (en) | 2003-01-22 | 2017-12-26 | Chr. Hansen A/S | Storage stable frozen lactic acid bacteria culture |
US20190223478A1 (en) * | 2016-07-15 | 2019-07-25 | Cj Cheiljedang Corporation | Leuconostoc mesenteroides cjlm627 strain producing reduced amount of gas, and kimchi production method using same |
US20190297908A1 (en) * | 2016-07-15 | 2019-10-03 | Cj Cheiljedang Corporation | Leuconostoc mesenteroides cjlm119 strain producing reduced amount of gas, and kimchi production method using same |
US10624355B2 (en) | 2013-08-20 | 2020-04-21 | Lallemand, Inc. | Modern preferment method for manufacturing dough mixture |
US11426345B2 (en) | 2015-01-27 | 2022-08-30 | The Johns Hopkins University | Hypotonic hydrogel formulations for enhanced transport of active agents at mucosal surfaces |
CN115558620A (en) * | 2022-09-30 | 2023-01-03 | 微康益生菌(苏州)股份有限公司 | Lactic acid bacteria freeze-drying protective agent and preparation method and application thereof |
US12005089B2 (en) * | 2014-12-15 | 2024-06-11 | The Johns Hopkins University | CVS transplantation for treatment of bacterial vaginosis |
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US8481027B2 (en) | 2003-01-22 | 2013-07-09 | Chr. Hansen A/S | Frozen lactic acid bacterial culture of individual pellet |
ATE466070T1 (en) * | 2004-02-24 | 2010-05-15 | Chr Hansen As | FROZEN GRANULE-FORMING LACTIC ACID BACTERIA CULTURE |
DE102005003457B4 (en) * | 2004-07-30 | 2009-12-03 | IfP Privates Institut für Produktqualität GmbH | Method and parts set for the microbiological determination of vitamines |
EP1789529B1 (en) | 2004-08-20 | 2013-10-02 | Actogenix N.V. | Method to improve lactococcus preservation |
ITMI20062287A1 (en) * | 2006-11-28 | 2008-05-29 | Anidral Srl | METHOD FOR THE PREPARATION OF BACTERIAL CROPS FROZEN FOR USE IN FIELD OF DIETARY NUTRACEUTICAL AND PHARMACEUTICAL FOOD |
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DK2424972T3 (en) * | 2009-04-30 | 2013-10-14 | Actogenix Nv | Cryoprotectants for freeze-drying of lactic acid bacteria |
RU2585846C2 (en) | 2010-04-27 | 2016-06-10 | Кр. Хансен А/С | Method for inoculating yeast into fruit juice |
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EP2730646B2 (en) | 2011-06-30 | 2020-01-22 | Chr. Hansen A/S | Drying lactic acid bacteria and new compositions |
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US20140348978A1 (en) | 2011-12-23 | 2014-11-27 | Chr. Hansen A/S | Method for making cheese |
CN103380887A (en) * | 2012-05-06 | 2013-11-06 | 黑龙江大荒春酒业有限公司 | Vegetable leavening agent and preparation method thereof |
JP2021521863A (en) * | 2018-05-04 | 2021-08-30 | セーホーエル.ハンセン アクティーゼルスカブ | Recovery and improvement of nitrate reductase activity |
AR120265A1 (en) | 2019-10-23 | 2022-02-09 | Chr Hansen As | BACTERIAL COMPOSITION TO CONTROL FUNGUS DETERIORATION AND ITS USE |
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US4262023A (en) * | 1977-12-22 | 1981-04-14 | Mauri Brothers & Thomson (Aust.) Pty. Limited | Frozen multiple-strain cheese starter composition |
US5637494A (en) * | 1990-01-29 | 1997-06-10 | Ecosyl Products Ltd. | Stabilized cultures of microorganisms |
US5677166A (en) * | 1995-06-05 | 1997-10-14 | Utah State University, Office Of Technology Commercialization | Compositions and methods for phage resistance in dairy fermentations |
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NL175193C (en) | 1972-05-02 | 1984-10-01 | Stichting Bedrijven Van Het | METHOD FOR PREPARING A CULTURE OF LACTIC ACID BACTERIA. |
DK60677A (en) | 1977-02-11 | 1978-08-12 | Hansen S Labatorium C | FREEZER |
US4140800A (en) * | 1977-06-13 | 1979-02-20 | Leo Kline | Freeze-dried natural sour dough starter |
EP0259739A1 (en) | 1986-09-10 | 1988-03-16 | Rhone-Poulenc Inc. | Improved stability of freeze-dried cultures |
AU1773700A (en) * | 1998-08-26 | 2000-07-31 | Chr. Hansen A/S | Liquid starter cultures having improved storage stability and use thereof |
US8481027B2 (en) | 2003-01-22 | 2013-07-09 | Chr. Hansen A/S | Frozen lactic acid bacterial culture of individual pellet |
EP1441027A1 (en) | 2003-01-22 | 2004-07-28 | Chr. Hansen A/S | Storage stable frozen lactic acid bacteria |
ATE466070T1 (en) | 2004-02-24 | 2010-05-15 | Chr Hansen As | FROZEN GRANULE-FORMING LACTIC ACID BACTERIA CULTURE |
-
2003
- 2003-01-22 EP EP03001442A patent/EP1441027A1/en not_active Withdrawn
-
2004
- 2004-01-19 DK DK04703147.1T patent/DK1587909T3/en active
- 2004-01-19 US US10/543,240 patent/US20060204484A1/en not_active Abandoned
- 2004-01-19 WO PCT/DK2004/000025 patent/WO2004065584A1/en active Application Filing
- 2004-01-19 ES ES04703147T patent/ES2427542T3/en not_active Expired - Lifetime
- 2004-01-19 EP EP04703147.1A patent/EP1587909B1/en not_active Revoked
-
2014
- 2014-12-31 US US14/587,953 patent/US9848615B2/en not_active Expired - Lifetime
Patent Citations (7)
Publication number | Priority date | Publication date | Assignee | Title |
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US4180800A (en) * | 1977-07-07 | 1979-12-25 | Sumitomo Electric Industries, Ltd. | Character reading system |
US4262023A (en) * | 1977-12-22 | 1981-04-14 | Mauri Brothers & Thomson (Aust.) Pty. Limited | Frozen multiple-strain cheese starter composition |
US5637494A (en) * | 1990-01-29 | 1997-06-10 | Ecosyl Products Ltd. | Stabilized cultures of microorganisms |
US5677166A (en) * | 1995-06-05 | 1997-10-14 | Utah State University, Office Of Technology Commercialization | Compositions and methods for phage resistance in dairy fermentations |
US5919695A (en) * | 1997-04-22 | 1999-07-06 | Quest International, B.V. | Method and culture for inhibiting undesired microorganisms with culture of Bacillus subtilis |
US20020054936A1 (en) * | 1997-08-25 | 2002-05-09 | Chr. Hansen A/S | Dairy starter culture delivery system and use hereof |
US20020031527A1 (en) * | 1998-11-16 | 2002-03-14 | Introgen Therapeutics, Inc. | Formulation of adenovirus for gene therapy |
Cited By (13)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US9848615B2 (en) | 2003-01-22 | 2017-12-26 | Chr. Hansen A/S | Storage stable frozen lactic acid bacteria culture |
US20110020494A1 (en) * | 2007-10-30 | 2011-01-27 | Lars Petersen | Growth medium for lactic acid bacteria |
US20120003721A1 (en) * | 2009-02-23 | 2012-01-05 | Birgitte Yde | Method for making lactic acid bacteria composition |
US8501456B2 (en) * | 2009-02-23 | 2013-08-06 | Chr. Hansen A/S | Method for making lactic acid bacteria composition |
US10370636B2 (en) | 2010-12-10 | 2019-08-06 | Dsm Ip Assets B.V. | Starter culture compositions |
EP2649175B1 (en) | 2010-12-10 | 2017-11-29 | DSM IP Assets B.V. | Starter culture compositions |
US10624355B2 (en) | 2013-08-20 | 2020-04-21 | Lallemand, Inc. | Modern preferment method for manufacturing dough mixture |
US11633350B2 (en) | 2014-02-23 | 2023-04-25 | The Johns Hopkins University | Hypotonic microbicidal formulations and methods of use |
US12005089B2 (en) * | 2014-12-15 | 2024-06-11 | The Johns Hopkins University | CVS transplantation for treatment of bacterial vaginosis |
US11426345B2 (en) | 2015-01-27 | 2022-08-30 | The Johns Hopkins University | Hypotonic hydrogel formulations for enhanced transport of active agents at mucosal surfaces |
US20190223478A1 (en) * | 2016-07-15 | 2019-07-25 | Cj Cheiljedang Corporation | Leuconostoc mesenteroides cjlm627 strain producing reduced amount of gas, and kimchi production method using same |
US20190297908A1 (en) * | 2016-07-15 | 2019-10-03 | Cj Cheiljedang Corporation | Leuconostoc mesenteroides cjlm119 strain producing reduced amount of gas, and kimchi production method using same |
CN115558620A (en) * | 2022-09-30 | 2023-01-03 | 微康益生菌(苏州)股份有限公司 | Lactic acid bacteria freeze-drying protective agent and preparation method and application thereof |
Also Published As
Publication number | Publication date |
---|---|
EP1441027A1 (en) | 2004-07-28 |
EP1587909A1 (en) | 2005-10-26 |
DK1587909T3 (en) | 2013-10-14 |
EP1587909B1 (en) | 2013-06-19 |
WO2004065584A8 (en) | 2004-10-14 |
ES2427542T3 (en) | 2013-10-30 |
US9848615B2 (en) | 2017-12-26 |
US20150223480A1 (en) | 2015-08-13 |
WO2004065584A1 (en) | 2004-08-05 |
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Legal Events
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STCB | Information on status: application discontinuation |
Free format text: ABANDONED -- AFTER EXAMINER'S ANSWER OR BOARD OF APPEALS DECISION |