AU2002300666A1 - Cryoprotection - Google Patents

Description

P/00/011 Regulation 3.2

AUSTRALIA

Patents Act 1990 COMPLETE SPECIFICATION FOR A STANDARD PATENT

ORIGINAL

TO BE COMPLETED BY APPLICANT Name of Applicant: Actual Inventors: Address for Service: Invention Title: THE UNIVERSITY OF NEWCASTLE RESEARCH ASSOCIATES (TUNRA) LTD.

CLAIRE NICOLE HEENAN, MICHELLE CATHERINE

ADAMS

CALLINAN LAWRIE, 711 High Street, Kew, Victoria 3101, Australia

CRYOPROTECTION

The following statement is a full description of this invention, including the best method of performing it known to us:- 30/07/02,mc12215.cs,1

CRYOPROTECTION

Technical Field This invention relates to the protection of microorganisms against lethal and sublethal damage caused by exposure to low temperatures. This protection is of use in frozen microorganisms and freeze drying microorganisms and in the preparation and storage of fermented and probiotic foods.

Background Art Microorganisms find application as starter cultures in the production of fermented foods, or as probiotic microorganisms providing health benefits to the consumer. Starter cultures and probiotic organisms can be: i) used for fermentation of raw ingredients; or ii) probiotic microorganisms can be added in suitable concentrations to the finished product prior to packaging (Bullimore, 1983; Gilliand, 1985). Starter and probiotic cultures can be preserved in frozen or freeze dried forms that can be either inoculated into the bulk starter media or directly into the product prior to fermentation or packaging.

Frozen starter cultures are the least expensive to produce but do require a continuous cold chain from place of manufacture to the point of use. Further to this, long term storage of frozen blocks is best below -20'C, temperatures that might not be easily attainable in the factory. Freeze dried starter cultures are more expensive to produce but have the added advantage of being able to be transported without refrigeration (Champagne et al., 1991). Consequently, freeze dried cultures are a better alternative for the food manufacturer that does not possess the facilities to produce and store its own cultures. Spray dried lactic acid bacteria cultures are still in an experimental stage and good cell viability is not yet standard (To and Etzel, 1997).

Freezing and freeze drying microorganisms can affect their viability.

Cryoprotectants are compounds that provide some protection to biological materials during freezing, freeze drying and subsequent storage. These compounds are used to prevent lethal and sub-lethal damage from occurring to the microorganisms, in order to maintain maximum viability, metabolic activity or health benefits in subsequent applications of the microorganisms.

Many compounds have been trialed as cryoprotectants. Cryoprotectants including skim milk; disaccharides: lactose, sucrose and trehalose; polyols: glycerol, adonitol, sorbitol; polysaccharides: pectin, dextran, resistant starch; amino acids; polymers: gelatin, 30/07/02,mcl 2215.speci,2 gums, maltodextrin; and antioxidants: ascorbic acid; have been trialed with mixed and often poor results (Champagne et al. 1991).The modes of action of cryoprotectants are not generally well understood. Cryoprotectant action is thought to be a combination of many factors including stabilization of microbial cell membranes, retention of high water activity and prevention of ice crystal formation, preventing oxidation, eradication of free radicals and prevention of subsequent cell disruption (Champagne et al., 1991).

Detrimental treatment of microorganisms during freeze drying, may result in reduction of viability, metabolic pathway damage, reduced fermentation activity or tolerance of adverse conditions. Changes in metabolic activity, pathogen inhibition, salt, bile and acid tolerance are indicative of the degree of damage sustained during freeze drying. The most important feature of a freeze dried culture is retention of cell viability.

After freeze drying, probiotic cultures should preferably retain both maximum viability and activity, or the specific attributes associated with that isolate.

For probiotic microorganisms to be of benefit to the consumer, they must be present in the food or tablets in a suitable concentration. When incorporated into a product, probiotics need to remain viable at a concentration of 10 6 cfu/gram or greater for the entire shelf life of the product to be of benefit to the consumer. A person would then need to consume at least 100 grams of the product every day to ensure a minimum daily dose ofprobiotics of 10 8 cfu in total, in order to gain health benefits.

Probiotic viability in food products has been shown to be a significant issue, as initial results revealed that many products, such as yoghurt, did not maintain adequate probiotic survival (Rybka and Fleet, 1997). Better survival of probiotic bacteria in yoghurts has been achieved by adding resistant starch to the yoghurt (CRC for Food Industry Innovation, 1997) Probiotic microorganisms incorporated into frozen fermented dairy yoghurt and ice cream products have shown better viability during shelf life when compared to chilled yoghurts. As well as being present in adequate concentrations, probiotics need to be in a condition suitable to cope with the adverse acid and bile conditions encountered in the gastrointestinal tract. Product manufacture may injure probiotic cells. Although they remain viable, impaired cells are readily inactivated if less than ideal conditions are experienced, for example, on exposure to acid conditions of the stomach and bile salts of the gastrointestinal tract. Sublethal injury can be assessed by measuring changes in normal cell activity or cell resistance, such as 1-galactosidase activity or bile tolerance.

30/07/02,mcl 2215.speci,3 -4- Description of the Invention The present inventors investigated the use of various substances as cryoprotectants for microorganisms used in the preparation of, or incorporated into, foods.

The cryoprotectant which is currently typically used is skim milk. One problem with skim milk is that its animal origin makes its use unacceptable to strict vegetarians.

Trehalose has also been used but it is an expensive compound which detracts from its use in relation to foods.

Inulin is a plant-derived oligo/polysaccharide comprising branched glucose and fructose chains of heterogeneous lengths with the chemical structure of ca-D-Glu-(1-2)-[(p- D-Fru-(1-2)-] n (Crittenden, 1999). Inulin, marketed as Raftiline" (Orafti, Aandorenstraat 1, 3300 Tienen, Belgium), is non-digestible to humans, but acts as a 'prebiotic', selectively being utilised by Bifidobacterium species in the human gut (Roberfroid, 1993).

The information supplied by the marketing company reports that the strain B. lactis Bb-12 can utilise inulin although the test conditions are not mentioned. Inulin has a low calorific content and also acts as soluble dietary fibre. Inulin is already used in foods intended as dietary aids, with fibre, fat replacer and improved textural qualities.

The present inventors included the prebiotic inulin in the substances they tested as cryoprotectants reasoning that if a substance reported to function as a prebiotic proved to have cryoprotective properties it would be particularly beneficial to the production of probiotic foods, especially, in the case of inulin, those acceptable to vegetarians.

The present inventors surprisingly found that inulin was not only cryoprotective but could provide better cryoprotection than skim milk as illustrated by providing better survival of microorganisms during freeze drying.

The present invention provides use of an oligo/polysaccharide comprising branched glucose and fructose chains of heterogeneous lengths as a cryoprotectant for microorganisms. Typically the oligo/polysaccharide is of plant origin. Plants which can act as a source of such an oligo/polysaccharide include topinambour, chicory, onion, asparagus and artichoke.

In particular the present invention provides use of inulin as a cryoprotectant for microorganisms.

The present invention provides a method for freeze drying a microorganism, which method comprises freeze drying the microorganism in the presence of an oligo/polysaccharide comprising branched glucose and fructose chains of heterogeneous lengths.

30/0 7 /02,mcl221 5 .speci,4 The present invention provides a method for freeze drying a microorganism, which method comprises freeze drying the microorganism in the presence of inulin.

The present invention provides a method for freezing a microorganism, which method comprises freezing the microorganism in the presence of an oligo/polysaccharide comprising branched glucose and fructose chains of heterogeneous lengths.

The present invention provides a method for freezing a microorganism, which method comprises freezing the microorganism in the presence of inulin.

The invention provides a method for preventing cell deactivation during freeze drying of a microorganism which method comprises freeze drying the microorganism in the presence of an oligo/polysaccharide comprising branched glucose and fructose chains of heterogeneous lengths.

The invention provides a method for preventing cell deactivation during freeze drying of a microorganism which method comprises freeze drying the microorganism in the presence of inulin.

The invention provides a method for preventing cell deactivation during freezing of a microorganism which method comprises freezing the microorganism in the presence of an oligo/polysaccharide comprising branched glucose and fructose chains of heterogeneous lengths.

The invention provides a method for preventing cell deactivation during freezing of a microorganism which method comprises freezing the microorganism in the presence of inulin.

The invention provides a method for preventing sublethal injury during freeze drying of a microorganism, which method comprises freeze drying the microorganism in the presence of an oligo/polysaccharide comprising branched glucose and fructose chains of heterogeneous lengths.

The invention provides a method for preventing sublethal injury during freeze drying of a microorganism, which method comprises freeze drying the microorganism in the presence of inulin.

The invention provides a method for preventing sublethal injury during freezing of a microorganism, which method comprises freezing the microorganism in the presence of an oligo/polysaccharide comprising branched glucose and fructose chains of heterogeneous lengths.

30/07/02,mcl 2215.speci,5 -6- The invention provides a method for preventing sublethal injury during freezing of a microorganism, which method comprises freezing the microorganism in the presence of inulin.

The invention provides a method for enhancing storage survival of a microorganism which method comprises freeze drying the microorganism in the presence of an oligo/polysaccharide comprising branched glucose and fructose chains of heterogeneous lengths.

The invention provides a method for enhancing storage survival of a microorganism which method comprises freeze drying the microorganism in the presence of inulin.

The invention provides a method for enhancing storage survival of a microorganism which method comprises freezing the microorganism in the presence of an oligo/polysaccharide comprising branched glucose and fructose chains of heterogeneous lengths.

The invention provides a method for enhancing storage survival of a microorganism which method comprises freezing the microorganism in the presence of inulin.

The invention provides a method for enhancing storage survival of a microorganism which method comprises storing the microorganism in the presence of an oligo/polysaccharide comprising branched glucose and fructose chains of heterogeneous lengths.

The invention provides a method for enhancing storage survival of a microorganism which method comprises storing the microorganism in the presence of inulin.

The present invention provides a culture of a microorganism which has been prepared using one or more methods of the present invention.

The microorganism is particularly selected from a microorganism used in the preparation of a food and a probiotic microorganism.

The present invention also provides a food incorporating one or more microorganisms prepared by a method of the invention.

The present invention also provides a food prepared using one or more microorganisms prepared by a method of the invention.

In one aspect the present invention provides a cold food incorporating one or more microorganisms prepared by a method of the invention. The cold food is a food which is 30/07/02,mcl 2215.speci,6 maintained at a temperature below room temperature but above freezing. Typically the storage temperature is about 4 0

C.

In another aspect the present invention provides a frozen food incorporating one or more microorganisms prepared by a method of the invention. The frozen food is a food which is maintained at a temperature below its freezing point. Typically the storage temperature is about -20 0

C.

In another aspect the present invention provides a cold food prepared using one or more microorganisms prepared by a method of the invention. The cold food is a food which is maintained at a temperature below room temperature but above freezing.

Typically the storage temperature is about 4 0

C.

In another aspect the present invention provides a frozen food prepared using one or more microorganisms prepared by a method of the invention. The frozen food is a food which is maintained at a temperature below its freezing point. Typically the storage temperature is about In yet another aspect the present invention provides a vegetarian food incorporating one or more microorganisms prepared by a method of the invention. The vegetarian food may be a cold food or a frozen food.

In yet another aspect the present invention provides a vegetarian food prepared using one or more microorganisms prepared by a method of the invention. The vegetarian food may be a cold food or a frozen food.

The food may be prepared by adding the one or more microorganisms to the already prepared food.

Alternatively, the one or more microorganisms may be added to the food during preparation. This may result in growth of the microorganism(s) within the food and resultant effects on the properties of the food. The microorganism(s) may participate in fermentation of raw materials in the preparation. The microorganism(s) may provide partial or complete fermentation of raw materials in the preparation. The microorganisms used in this way may only provide fermentative functions or may provide fermentation and probiotic functions.

The food may be prepared using one or more microorganisms and then have further microorganisms added.

The present invention provides a method for extending the shelf life of a food containing one or more microorganisms which method comprises incorporating an 30/07/02,mcl 2215.speci,7 oligo/polysaccharide comprising branched glucose and fructose chains of heterogeneous lengths in the food.

The present invention provides a method for extending the shelf life of a food containing one or more microorganisms which method comprises incorporating inulin in the food.

The present invention provides a method for increasing the survival of one or microorganisms in a food or health supplement which method comprises incorporating an oligo/polysaccharide comprising branched glucose and fructose chains of heterogeneous lengths in the food or supplement.

The microorganism(s) may be prepared in capsular form with the oligo/polysaccharide comprising branched glucose and fructose chains of heterogeneous lengths present in the encapsulated form. The microorganism(s) in this form may be used as a health supplement or may in turn be incorporated into a food.

The microorganism(s) may also be prepared in tablet form with the oligo/polysaccharide comprising branched glucose and fructose chains of heterogeneous lengths present in the tablet. The microorganism(s) in this form may be used as a health supplement or may in turn be incorporated into a food.

The present invention provides a method for increasing the survival of one or microorganisms in a food or health supplement which method comprises incorporating inulin in the food or supplement.

The microorganism(s) may be prepared in capsular form with inulin present in the encapsulated form. The microorganism(s) in this form may be used as a health supplement or may in turn be incorporated into a food. The microorganism(s) may be prepared in tablet form with inulin present in the tablet. The microorganism(s) in this form may be used as a health supplement or incorporated into a food.

The present invention encompasses the use of all fermentative and probiotic microorganisms acceptable for food production for humans or animals.

Utilisation of commercially available isolates ensures the continuing availability of the strain; secondly, the strains are already guaranteed to be safe for consumption; and for probiotic microorganisms ensures they will survive the passage through the gastrointestinal tract, and as long as they are present in sufficient numbers, will confer their particular health benefits to the consumer.

Lactic acid bacteria and probiotic cultures for commercial food production, and starter cultures for fermentation can be purchased from international companies such as 30/07/02,mcl 2215.speci,8 Christian Hansen Pty Ltd (Bayswater, Australia) and Gist-Brocades Australia Pty Ltd (Moorebank, Australia). Other research organisations such as the CSIRO Starter Culture Collection (Highett, Australia) and the Australian Starter Culture Research Centre (Werribee, Australia), have good lactic acid bacteria and potential probiotic collections but these organisms are only available on a small scale.

Table 1 Species currently used as probiotics around the world.

Lactobacillus spp. Bifidobacterium spp.

L. acidophilus B. bifidum L. johnsonii B. longum L. paracasei ssp. Paracasei B. infantis L. rhamnosus B. breve L. plantarum B. adolescentis L. brevis B. lactis L. reuteri L. salivarius L. fermentum L. helveticus L. delbrueckii ssp. Bulgaricus Other Species Streptococcus salivarius ssp. Thermophilus Lactococcus lactis ssp. Lactis and cremoris Enterococcus faecium Leuconostoc mesenteroides ssp. Dextranium Propionibacterium freudenreichii Pediococcus acidilactici Saccharomyces boulardii Escherichia coli Bacteroides spp.

Bacillus spp.

Adapted from O'Sullivan et al. (1992), Sanders (1999) and Rolfe (2000) Selection criteria for probiotic isolates There is no consensus on how to define or accredit a microorganism as probiotic (Guarner and Schaafsma, 1998). Each strain that is considered for use in human probiotic preparations needs to be subjected to strict characterisation and experimentation to ensure 3 0/07/02,mcl 2215.speci,9 the effectiveness and safety of the particular strain. Table 2 lists the criteria for assessing potential probiotic strains.

Characterisation tests for assessing potential probiotic microorganisms for human use are designed to select the most appropriate strains and ensure the efficacy of the product. As all probiotic strains do not cover the complete range of health benefits, specific targets should be identified when selecting a strain.

Table 2 Requirements for good clinical studies demonstrating unique probiotic properties for functional food use.

Each specimen and each strain should be documented and tested independently, on their own merit Extrapolation of data from closely related strains is not acceptable Well-defined probiotic species and strains, with well-described study preparations Double-blind, placebo-controlled human studies Randomised human studies Results confirmed by different independent research groups Publication in respectable international peer-reviewed journals Taken from Salminen and Saxelin (1996).

Table 3 Desirable criteria for selecting a probiotic strain for human use.

Probiotic strain must be of host origin and properly identified The strain must be clinically safe for use in foods, with no side effects Exhibit stable characteristics in storage and in foods Be industrially compatible able to be grown to desired concentration, suitable taste qualities, survive production Survive on route to the large intestine acid, bile and lysozyme resistant Colonised or adhere to the gastrointestinal tract Exhibit demonstrable health benefits improved nutritional value, prevention of diarrhoea and constipation, pathogen inhibition, immune system stimulation and modulation, cancer prevention, modulate metabolic activities 30/0 7 /02,mcl 221 5.speci, -11- Adapted from Klaenhammer and Kullen (1999) and Gibson and Fuller (2000) Host origin Probiotic microorganisms should be of human origin if they are intended for human consumption. Not only is this a safety consideration in terms of cross-species pathogenicity, but also strains that have been repeatedly isolated from humans are more likely to adapted to that ecosystem and stand a better chance of survival. Microorganisms isolated from other ecological systems may prove to be pathogenic or at least undesirable when consumed by humans.

Isolates intended for use in other animal species typically originate from the species in which they are intended to be used Safety Safety in consuming live cultures is one of the most important factors when examining potential probiotic microorganisms. Lactobacillus (with the exception of L.

rhamnosus), Bifidobacterium and S. boulardii are not considered to pose a risk to consumers, however every new strain should be examined for safety aspects. Donohue and Salminen (1996) suggested a set of criteria that potential probiotics should satisfy to ensure safety when consumed by humans.

Table 4 Suitable models and methods to test the safety of potential probiotic strains for human consumption.

1. Determine the intrinsic properties of the strain antibiotic resistance, plasmid transfers etc.

2. Assess the effects of the metabolic products from the microorganism 3. Assess the acute and subacute toxicity of ingesting large amounts of the microorganism 4. Estimate in vitro infective properties in cell culture and then in animal models Determine the efficacy of ingested probiotic by dose response and impact on the composition of human intestinal microflora 6. Identify and assess any side effects in human trials 7. Epidemiological surveillance of people consuming the new introduced probiotic 8. The most rigorous safety testing for genetically modified or animal derived strains 30/07/02,mcl 2215.speci,11 -12- Taken from Donohue and Salminen (1996) Survival in the gastrointestinal tract When consumed, probiotic microorganisms have to survive the passage from the mouth, through the stomach, to the intestines to exert any influence on the host.

Bifidobacteria are reported to be predominantly located in the caecum, whereas Lactobacillus species preferably colonise the ileum. For best survival rates, strains need to be acid, bile and lysozyme tolerant to provide a competitive advantage in vivo. Bile and acid resistance in probiotic microorganisms has been trialed in vitro, using batch and multiple chemostat techniques, with media containing appropriate levels of bile or buffered at low pH to mimic the gastrointestinal system. In vitro experiments are not ideal, but do effectively highlight inadequate species and provide a starting point for further experimentation.

Intestinal adhesion or colonisation Ideally, it is desirable that probiotic microorganisms adhere to or colonise the intestine. The benefits of colonisation include displacement or exclusion of undesirable bacteria or pathogens from adhering to the intestines and prolonged existence of desirable bacteria in the intestines. The longer probiotic microorganisms are present in the intestines, the greater any possibility of beneficial effect on the host.

Although colonisation does not appear to be permanent, delayed residence in the large intestine is apparent with some strains. After feeding has ceased, many probiotic strains can be isolated from faeces for days or weeks afterwards before dying out. Sanders (Sanders, 1993) speculates that although probiotics are not permanent residents, continuous consumption of these transient probiotic organisms does appear to be a requirement for prolonged health benefits.

Many pathogens rely on adhesion to the intestinal mucosa as the first stage of host infection and probiotic microorganisms that prevent this initial step would be beneficial.

Bernet et al. (1993) showed that several species ofBifidobacterium could inhibit cell adhesion and invasion by E. coli and S. typhimurium. Other similar in vitro cell culture experiments have been conducted, all reporting strain specific adhesion. Out of 12 Lactobacillus strains only 4 strains, L. casei 744, L. acidophilus Lal, L. rhamnosus LC- 705 and L. rhamnosus GG, displayed significantly greater adherence than the non specific binding of E. coli.

30/07/02,mcl 2215.speci,12 13- Industrial exploitation For industrial food production, a probiotic strain must be technologically exploitable. It is important that the bacterium be grown easily and be able to withstand food processing. Additional hours incubating a slow growing bacterium adds to the cost of production. Probiotic species need to maintain stable characteristics during production, short and long term storage and the shelf life of the food product. Rapid acidification of fermented foods by lactic acid bacteria is required to inhibit pathogens as well as impart organoleptic qualities. Strain stability also includes retaining the traits associated with health benefits for the host, such as bile and acid tolerance, and pathogen inhibition.

Survival in food systems Probiotic strains are isolated from and selected for their ability to survive in the gastrointestinal tract and consequently many species show poor survival in foods. L.

delbrueckii ssp. bulgaricus and Streptococcus thermophilus do not survive in the gastrointestinal tract environment, but they do have excellent industrial properties. Good manufacturing and survival characteristics are not inherent in all lactic acid bacteria, indicating that strain selection is an important factor for product manufacture.

The question of the required concentration of viable probiotic organisms is still unresolved. Viability is assumed to be related to activity and imparting health benefits, although this is not always the case. The consumption of probiotics at a level of 108 10 9 cfu per day is a commonly quoted figure for adequate probiotic consumption, equating to 100g of a food product containing 106 107 cfu/g.

The form in which probiotic bacteria are fed, affects the minimum dose for detection in the faeces. L. rhamnosus GG could be detected in host faeces when fed at a lower concentration of 10 9 cfu in fermented or sweet milk and as enterocapsules, rather than at 10 'cfu in a freeze dried capsules. The difference in survival was attributed to the milk buffering capacity and insoluble enterocapsule capsule coating, providing protection during transit through the stomach.

The final product should contain probiotic microorganisms at an adequate concentration for the entire shelf life of the product. The Australian Food Standard Code (Standard H8) stipulates that yoghurt must have a pH less than 4.5 and be prepared with S.

thermophilus and L. delbrueckii ssp. bulgaricus or other suitable cultures, but does not stipulate required levels of probiotic bacteria. Some countries have imposed loose standards of minimum allowable levels of probiotics or lactic acid bacteria in yoghurts.

30/07/02,mcl 2215.speci,13 14- There appear to be no specifications with regard to probiotic products that are not of dairy origin.

Probiotic survival in products is affected by a range of factors including pH, post acidification, hydrogen peroxide production, storage temperature, the mixture of starter cultures, packaging and food ingredients. Bifidobacteria and L. acidophilus show better survival when supplied with complex carbohydrates or oligosaccharides. Probiotic survival is generally better in mild acidic conditions, when the pH is above 4.

Tablets and capsules Tablets and capsules are prepared in accordance with standard techniques used in the health industry for their preparation.

The inulin, used by the present inventors was a high purity inulin gel which was sterilised with heat prior to use. The present invention relates to use of inulin in this form but also relates to use of other forms of inulin whether heat sterilised or not. Similarly, the present invention relates to use of an oligo/polysaccharide comprising branched glucose and fructose chains of heterogeneous lengths as a high purity gel which is sterilised with heat prior to use. The present invention also relates to use of other forms of the oligo/polysaccharide whether heat sterilised or not. The desirability of sterilising materials for use in growing or storing microorganisms will be self evident to the skilled addressee, as will be the fact that sterilisation can be carried out in other ways.

Definitions Oligosaccharide: a glycoside containing between three and ten sugar moieties Polysaccharide: a glycoside containing between three and eighty sugar moieties Comprising: where the terms "comprise", "comprises", "comprised" or "comprising" are used in this specification, they are to be interpreted as specifying the presence of the stated features, integers, steps or components referred to, but not to preclude the presence or addition of one or more other features, integers, steps, components or groups thereof Cryoprotectant: a cryoprotectant is any compound that protects biological material or cells from the detrimental effects of cold temperatures in preparation or storage.

3 0/0 7 /02,mcl 2215.speci,14 Brief Description of the Drawings Figure 1 shows the expected and observed cell concentrations after freeze drying in various cryoprotectants, for probiotic bacteria grown in SPY 2 and SPY 6.L. acidophilus MJLA1 grown in a) SPY 2, b) SPY 6; L. rhamnosus LCSH1 grown in c) SPY 2, d) SPY 6; and B. lactis BDBB2 grown in e) SPY 2, f) SPY 6.

Figure 2 shows the angle of decline of viability and bile tolerance during storage of various probiotic organisms freeze dried in different cryoprotectants: a) L. acidophilus MJLA1, L. rhamnosus LCSH1 and c) B. lactis BDBB2 grown in SPY 2 (solid circles) and SPY 6 (solid triangles) when recovered on agar (open symbols) and agar 0.3% bile (closed symbols).

Figure 3 shows the inhibition by L. acidophilus MJLA1 ofE. coli after growth in a) SPY 2, or b) SPY 6, and L. monocytogenes after growth in c) SPY 2, d) SPY 6 and freeze drying in various cryoprotectants.

Figure 4 shows the inhibition by L. rhamnosus LCSH1 ofE. coli after growth in a) SPY 2, or b) SPY 6, and L. monocytogenes after growth in c) SPY 2, d) SPY 6 and freeze drying in various cryoprotectants.

Figure 5 shows the inhibition by B. lactis BDBB2 ofE. coli after growth in a) SPY 2, or b) SPY 6, and L. monocytogenes after growth in c) SPY 2, d) SPY 6 and freeze drying in various cryoprotectants.

Figure 6 shows the acidification activity of probiotic organisms after freeze drying in various cryoprotectants: L. acidophilus MJLA1 grown in a) SPY 2, b) SPY 6; L.

rhamnosus LCSH1 grown in c) SPY 2, d) SPY 6; and B. lactis BDBB2 grown in e) SPY 2, f) SPY 6 and freeze dried in cryoprotectants.

Best Method of Carrying Out the Invention The present invention provides a cryoprotectant suitable for use in freeze drying microorganisms and in cold or frozen foods containing probiotic microorganisms. The cryoprotectant does not contain any animal-derived ingredients, and produces excellent cell viability and retention of probiotic characteristics. The cryoprotectant acts as a 30/0 7 /02,mcl 2215.speci,15 -16replacement for non-fat skim milk (NFSM), the most commonly used cryoprotectant. The use of NFSM as a cryoprotectant makes subsequent use of the microorganisms unsuitable for consumption by vegetarians or people with milk allergy or hypersensitivity.

According to the literature, the ideal cryoprotectant needs to bind water, prevent ice crystal formation, protect cell membranes, enhance cell shielding, prevent oxidation and eradicate free radicals. No realistic replacement for skim milk as a cryoprotectant has been identified prior to the present invention.

Exogenous conditions that affect survival of freeze drying include method and time of cell harvest and correct storage of freeze dried powders. Cells should be harvested at early stationary phase. Viability declined sooner in cells harvested during late log phase during extended storage. Better recovery is achieved when cells had been harvested by filtration or ultrafiltration rather than centrifugation, however, better survival occurred in cells obtained by centrifugation or ultrafiltration. During centrifuging, a higher temperature aids cell separation, but temperatures around 5°C is less detrimental to cell viability.

Bozoglu et al. (1987) modelled the survival kinetics of lactic acid bacteria and concluded that cell death is related to the area exposed to the external conditions. The shielding effect can be optimised by reducing cell surface area exposed the external conditions by using 'small' cell variants and harvesting cells to concentration dense enough to be beneficial without causing osmotic problems, approximately 10 Once freeze dried, cells must be stored under the right conditions to maintain maximum viability. Water activity between 0.1- 0.2 has been shown to be best with over drying and under drying both detrimental to cell viability. The dried cultures should be kept under vacuum or nitrogen gas, but not air or oxygen gas, to prevent oxidation. The cells retain greater viability when stored at refrigerated temperatures of 5°C and below.

The packaging should be moisture proof, oxygen proof and opaque.

Materials and Methods Microorganisms Probiotic cultures Lactobacillus acidophilus MJLA1, Bifidobacterium lactis BDBB2, and Lactobacillus rhamnosus LCSH1, were supplied by Christian Hansen 30/07/02,mcl2215.speci,16 -17- (Bayswater, Victoria Australia) and used in freeze drying cryoprotectant experiments. L.

acidophilus MJLA1 was used in the cryoprotectant concentration and antioxidant trials.

Microbiological growth media SPY 2 medium was prepared by dissolving 2.5% soy peptone, 2.5% yeast extract, and 2.5% glucose monohydrate in distilled water. The pH was adjusted to 7.0 using HCI or NaOH, the medium dispensed into bottles and autoclaved at 121°C for 15 mins. SPY 6 was prepared by fortifying SPY 2 medium with 0.1% Tween 80 prior to autoclaving. The medium was dispensed and autoclaved at 121 0 C for 15 mins and then further supplemented with sterile CaCl 2 .2H 2 0 and MnCl 2 .4H 2 0, to a final concentration of mM each.

MRS medium: de Man, Rogosa and Sharpe medium (de Man et al., 1960) RCM medium: Reinforced Clostridial medium (Hirsch and Grinsted, 1954) TSA medium: Tryptone Soya Agar (Oxoid Australia) NB medium: Nutrient Broth (Oxoid Australia) PBS: Phosphate buffered saline, (NaCl 8.00g, KCI 0.20g, Na 2

HPO

4 1.44g, KH 2 PO4 0.24g, distilled water 1 litre, pH Example 1 Freeze Drying Preservation of Cultures Probiotic cell production and harvest Bacteria were grown in of SPY 2 and SPY 6 (1 Litre) for 24 hours at 37C.

Lactobacilli were incubated in 8% CO 2 and bifidobacteria incubated in aerobic atmospheres. Cells were harvested by centrifugation (7000 x g at 4 0 C, 6min) and resuspended in each cryoprotectant (50mL) (Table to give an approximate concentration of 1010 cfu/mL. Cell suspensions were then frozen in a thin film, coating the inside of sterile conical flasks by rotating the flasks in dry ice. The frozen cell suspensions were then hardened at -80°C for 1 hour and then freeze dried -1.8 mbar, -40 0 Freeze dried powders were equilibrated to a water activity of 0. 1 by exposure to a saturated solution of lithium chloride in a sealed chamber for 48 hours. Dried cell suspensions were then gently aseptically ground, placed in pre-weighed specimen containers, reweighed, and 30/07/02,mcl 2215.speci,17 18stored in the dark, under vacuum mbar) at 5 0 C. Each experiment was conducted in triplicate.

Table 5 Compounds used as cryoprotectants for preserving various probiotic organisms during freeze-drying.

Cryoprotectant Sterilisation treatment Non-fat skim milk (Diploma) 10% pH 6.6, inspissated 100 0 C, Inulin HP-Gel (Orafti) 10% pH 6.3, inspissated 100'C, Yeast biomass (Sigma) 10% pH 7.0, inspissated 100°C, Trehalose (Sigma) 10% pH 7.0, autoclaved 121 0 C, Soy milk pH 7.0 UHT (Sanitarium Health Food Co, Australia) commercial package Soy protein isolate 5% pH 7.0, microfluidized at 7500 PSI, (IPT 545, International Protein Technologies) inspissated 100C, All cryoprotectant solutions were suspended in distilled water and sterilised as stated above. All cryoprotectants were used for microorganisms grown in SPY2, and skim milk, trehalose, inulin and yeast biomass were used for microorganisms grown in SPY 6.

Cell enumeration and bile sensitivity Cell viability and bile tolerance of the freeze dried cultures were assessed immediately prior to storage and at regular intervals over 6 months. Freeze dried powder (100mg, weighed accurately) was rehydrated with 0.1% peptone (2mL). The rehydrated cell slurry was serially diluted and plated on to MRS and MRS 0.3% for lactobacilli or RCA agar and RCA 0.3% bile (Oxoid) for bifidobacteria. Plates were incubated for 48 h at 37 0 C, lactobacilli at 8% CO 2 and bifidobacteria incubated anaerobically, after which the resulting colonies were counted. Cell concentrations were calculated as cfu/g powder.

Survival offreeze drying The theoretical maximum post freeze dried cell populations, the population in the freeze dried powder if no cells were deactivated, was calculated and compared to the actual viable counts in the freeze dried powder. Assuming that no losses were incurred during harvest or freeze drying, the theoretical maximum cell concentration per gram of freeze dried powder, is the number of organisms in the total volume of growth medium (cfu), divided by the final weight of the freeze dried powder the total colony forming 30/07/02,mcl2215.speci,18 -19units in 1 litre of SPY 2 or SPY 6 medium was calculated using maximum population data for each organism from previous growth medium trials.

Pathogen inhibition Bacteria were assessed for ability to inhibit E. coli (NCTC 11560) and Listeria monocytogenes (ATCC 7644) in vitro based on the methods of Chateau et al. (1993). Two aliquots (10 UL) of rehydrated cell suspension was spotted onto two MRS or RCA agar plates and incubated for 24h anaerobically to prevent H 2 0 2 accumulation. Plates were then overlayed with Tryptone Soya Agar (TSA; Oxoid) containing 0.1 mL of an overnight Nutrient Broth (Oxoid) culture of either E. coli or L. monocytogenes. Plates were incubated for 24 hours and the resulting zones of inhibition measured. The zone of inhibition was considered as the clear area between the edge of probiotic culture to edge of pathogen growth.

Acidification activity Each rehydrated cell slurry (0.2 mL) was inoculated into soy milk (2mL) (So Good, Sanitarium Health Food Co, Australia) that had been tempered to 37°C. The pH of the uninoculated and inoculated soy milk (pHinitial) was measured. The inoculated soy milk samples were then incubated at 37 0 C for 4 hours, the lactobacilli in 8% CO 2 and bifidobacteria anaerobically, after which the pH (pHfinal) was measured. Cell activity was calculated as change in pH per hour per log cfu/mL, using the following equation: Cell Activity ApH (pHinitial pH final) time (hours) x (Loglocfu/mL) Cryoprotectant concentration and antioxidants L. acidophilus strain MJLA1 was inoculated into SPY 6 medium (10mL) and incubated for 20 hours at 37 0 C in 8% CO 2 Cells were then harvested by centrifuging (5000 x g, 5 min), the supernatant discarded and the pellet resuspended in each cryoprotectant (1.OmL). Cryoprotectants used in this experiment are listed in An aliquot (0.8mL) of the resuspended cell concentrate was dispensed into an eppendorftube, frozen to -80C and then freeze dried overnight (-40 0 C, -1.8 mbar). The freeze dried samples were allowed to equilibrate to a water activity of 0. 1 by exposure to a saturated lithium 30/07/02,mcl2215.speci,19 chloride solution for 24 hours. Immediately after water activity equilibration, each tube was rehydrated to initial volume for immediate use in viability and activity tests. Each cryoprotectant was trialed in Table 6.

Table 6 Cryoprotectant suspension solutions for freeze-drying L. acidophilus strain MJLA1.

Cryoprotectant Treatment Control distilled H 2 0 Trehalose Trehalose Trehalose Inulin Inulin Inulin Trehalose inulin Trehalose inulin Trehalose inulin Trehalose tocopheral (Sigma) (10iL/L) Trehalose tocopheral (100L/L) Trehalose ascorbic acid (Sigma) (4mg/L) Trehalose ascorbic acid Inulin tocopheral (10pL/L) Inulin tocopheral (100 L/L) Inulin ascorbic acid (4mg/L) Inulin ascorbic acid pH 7.0, autoclaved 121°C, pH 7.0, autoclaved 121 0 C, pH 6.3, inspissated 100 0 C, pH 6.7, inspissated 100C, pH 7.0, trehalose solution autoclaved 121°C, 15mins. Sterile antioxidant added when cool.

pH 6.3, inulin solution inspissated 100 0 C, 15mins. Sterile antioxidant added when cool.

Cell viability and bile tolerance.

Prior to freeze drying and again immediately after rehydration, the viable cell populations and bile tolerant populations were enumerated by plate count using MRS and MRS 0.3% bile agar.

Acid tolerance Acid tolerance of each cell concentrate was assessed prior to freeze drying and immediately after rehydration. An aliquot of cell concentrate ImL) was mixed with phosphate buffered saline (1.0 mL, PBS, pH The cells were held for 3 hours at 37°C and then enumerated using MRS. The results were calculated as the number of acid tolerant bacteria present before and after freeze drying in the original cell concentrate.

30/07/02,mcl 2215.speci,20 -21

RESULTS

Probiotic survival during freeze drying The effect of freeze drying on cell viability varied depending on the cryoprotectant.

Figure 1 illustrates the actual viable and bile tolerant cell populations compared to the calculated theoretical maximum population after freeze drying.

When probiotic organisms were grown in SPY 2 medium, the least deactivation of cells occurred during freeze drying with trehalose and inulin as cryoprotectants (p<0.05).

Soy milk and skim milk were the next best cryoprotectants, followed by yeast biomass.

The highest initial deactivation occurred with soy protein as the cryoprotectant.

The degree of sub-lethal injury varied slightly between cryoprotectants, trehalose, yeast biomass, inulin and soy protein producing the least sub-lethal injury (p<0.05).

When the three strains of probiotic bacteria were grown in SPY 6, inulin gave the best protection to cells during freeze drying Trehalose was the next best, followed by skim milk and then yeast biomass. There were no significant differences between cryoprotectants with respect to sub-lethal injury of cells.

Survival offreeze dried probiotics during storage Freeze drying survival data were analysed by transforming the gradient of the 'line of best fit' to angle (degrees) from the x-axis. The greater the negative angle, the greater the decline in population. The angles were compared using ANOVA. The rate of cell deactivation during storage depended on the growth medium, cryoprotectant and the bacterium (p<0.05) When probiotic cells were cultured in SPY 2 (Figure the cryoprotectants that provided the best storage protection were trehalose and soy protein. Cell viability in these cryoprotectants and yeast biomass was better than when in skim milk. Trehalose and soy protein also provided the best stability of the bile tolerant population during storage, with the other cryoprotectants proving to be equally as good as each other.

When SPY 6 was used as a growth medium, of the four cryoprotectants used, trehalose, inulin, and skim milk were equally good in preserving cell viability and bile tolerance. Yeast biomass was not as effective in protecting cells 30/07/0 2 ,mcl 2215.speci,21 -22- Probiotic Inhibition of Pathogens The assessment of the ability of probiotic organisms to inhibit pathogens was conducted throughout the storage trial. The degree of inhibition of the pathogens was related to the cryoprotectant, the growth medium and the species of probiotic. The ability of probiotic organisms to inhibit pathogens did not obviously decline during the trial, but some probiotic species did display an erratic tendency on a weekly basis.

The cryoprotectants that gave the greatest inhibition were inulin and trehalose L. acidophilus MJLA1 (Figure 3) and L. rhamnosus LCSH1 (Figure 4) gave greater inhibition when they were grown in SPY 6 prior to freeze drying. Inhibition by B.

lactis BDBB2 (Figure 5) was not affected by growth medium.

Acidification activity The acidification activity per cell of freeze dried probiotic organisms during storage is presented in Figure 6.

When probiotic organisms were grown in SPY 2, using multi-factor ANOVA for data analysis, trehalose and inulin maintained the best acidification activity during storage Skim milk and soy milk were then next best, followed by soy protein, then yeast biomass. Probiotic organisms cultured in SPY 6 prior to freeze drying, retained the highest acidification activity in trehalose and skim milk Cells stored using yeast biomass as the cryoprotectant, had the biggest decrease in acidification ability during storage.

Overall, the decline in acidification activity ofprobiotic organisms grown in SPY 2 was lower than that of the organisms grown in SPY 6 However, there was no significant difference between cryoprotectants, using the results from both growth media.

The variations between bacteria and between media were too great.

Cryoprotectant concentration The effect of cryoprotectant concentration and the presence of antioxidants on cell viability, bile tolerance and acid tolerance during freeze drying were assessed. There were no significant differences in cell viability or bile tolerance due to cryoprotectant prior to freeze drying (Table There were differences in acid tolerance prior to freeze drying, depending on the cryoprotectant The acid tolerance treatment did decrease cell viability prior to and after freeze drying (p<0.01) (Table 8).

30/07/02,mcl 2215.speci,22 -23- Freeze drying caused a significant decrease in cell viability, bile tolerance and acid tolerance Water, the control, produced the lowest cell viability, bile and acid tolerance after freeze drying (p<0.05).

Inulin trehalose trehalose (7.5%)/inulin and trehalose ascorbic acid (4mg), provided the best protection to cell viability, bile and acid tolerance during freeze drying (p<0.05).

Ascorbic acid and tocopheral did not aid cell survival above that of 15% inulin and trehalose. The higher levels of antioxidants were generally detrimental to cell viability.

Table 7 Cell viability and bile tolerance ofL. acidophilus MJLA1 after freeze drying in various concentrations of cryoprotectants and antioxidants.

Cryoprotectant Water Trehalose Trehalose (10%) Trehalose (15%) Inulin Inulin (10%) Inulin (15%) Trehalose inulin Trehalose inulin Trehalose inulin Trehalose tocopheral (10L/L) Trehalose tocopheral (100tL/L) Trehalose ascorbic acid (4mg/L) Trehalose ascorbic acid (40mg/L) Inulin tocopheral (10L/L) Inulin tocopheral (100.L/L) Inulin ascorbic acid (4mg/L) Inulin ascorbic acid (40mg/L) Cell viability after freeze drying (log cfu/mL) mean s.d.

6.81 0.1 8.03c 0.0 8.20 abc 0.0 8.30a 0.0.

8.12 cd 0.1 8.18 a bc 0.0 8.29a 0.0 7.89e 0.0 8.04 d 0.0: 8.25 ab ±0.0 8.21 ab c 0.0 8.16 b c 0.0: 8.24 ab 0.0 8.16 bc ±0.0 8.10 c d 0.0 8.

1 6 bc 0.0: 8.20 a bc 0.1 8.14 bed 0.0 1 5 5 4 5 6 9 6 2 8 2 5 6 3 3 4 2 2 Bile tolerance after freeze drying (log cfu/mL) mean s.d.

5.79' 0.25 7.61 hg 0.05 8.07 ab 0.31 8.03 abc d 0.10 7.67 fgh 0.19 7.87 bcdef 0.08 8.13a 0.04 7.57 1 1 0.06 7.

8 1 efg 0.01 8.

0 4 abc 0.11 bcde 7.90 bc de 0.03 7.83de f 0.11 7.

9 3 a bcde 0.05 7.87 c def 0.07 7.

8 4 cdef 0.10 7.90 b de 0.02 7.

9 5 abc d e 0.11 7.

8 5 cdef 0.07 s.d. standard deviation etc significantly different survival compared to other cryoprotectants 30/0 7 /02,mcl 2215.speci,23 -24- Combinations oftrehalose and inulin were only successful at the highest concentration that was trialed. Trehalose (5%)/inulin and trehalose were not effective at maintaining cell viability, bile tolerance, or acid tolerance at reasonable levels.

Table 8 Acid tolerant cell populations before and after freeze drying in various concentrations of cryoprotectants and antioxidants.

Acid tolerance Acid tolerance after before freeze drying freeze drying Cryoprotectant (log cfu/mL) (log cfu/mL) mean s.d. Mean s.d Water 8.81 ab 0.05 6 2 7 g 0.08 Trehalose 8.71cd 0.04 7.

8 7 ef 0.20 Trehalose 8.68e 0.07 8.09be d 0.12 Trehalose 8 7 3 cd e 0.02 8.30a 0.08 Inulin 8 7 6 b de 0.03 8.

0 7 b c d 0.17 Inulin 8 7 9 abcd 0.12 8 0 9 bd 0.11 Inulin 8.

8 0 abed 0.05 8 2 2 a b 0.05 Trehalose inulin 8.

8 0 abc 0.06 7.72 f 0.12 Trehalose inulin 8 8 0 ab c d 0.01 7 9 6 de 0.02 Trehalose inulin 8 8 5 ab 0.05 8 1 6 abc 0.05 Trehalose tocopheral (10LL/L) 8 7 4 de 0.01 8.

0 4 c d 0.05 Trehalose tocopheral (100L/L) 8.

7 9 abcd 0.02 8 0 4 c d 0.03 Trehalose ascorbic acid (4mg/L) 8.85a 0.03 8.

14 abc 0.09 Trehalose ascorbic acid (40mg/L) 8 7 8 a bcd 0.03 8 .03 cde 0.08 Inulin tocopheral (10 tL/L) 8.67e 0.07 8 0 4 ed 0.02 Inulin tocopheral (100L/L) 8.75 cde 0.06 8 .03 cd e 0.03 Inulin ascorbic acid (4mg/L) 8.

7 8 abcd 0.07 8 12 bcd 0.15 Inulin ascorbic acid (40mg/L) 8.73ede 0.07 8 0 4 c d 0.02 s.d. standard deviation b et c significantly different survival compared to other cryoprotectants Discussion and Conclusions Culturing cells in SPY 2 and SPY 6 influenced the survival of organisms during freeze drying and subsequent storage, with SPY 6 producing the better results. Greater survival during freeze drying and a smaller population of sub-lethally injured cells was achieved by growing cells in SPY 6. Cells grown in SPY 6 also survived better during storage of the freeze dried cultures and retained a higher population of bile tolerant cells.

Adding Tween 80 and calcium to growth medium has been observed to improve survival, 30/07/02,mcl 2215.speci,24 without preventing sub-lethal injury. Probiotic bacteria cultured in SPY 2 did retain a higher degree of acidification ability during storage.

Freeze drying cryoprotectants As skim milk is the most commonly used cryoprotectant for freeze drying bacteria, it was used as a reference cryoprotectant, to which other cryoprotectants were compared in this experiment. The compounds trialed as cryoprotectants were trehalose, soy milk, inulin, yeast biomass and soy protein. Trehalose has been reported as a successful cryoprotectant in the past (Leslie, et al., 1995). Inulin and soy protein bind water very well and thus may retain a higher water content for the same water activity in a freeze dried state and reduce water stress. Soy milk is a dairy mimetic containing protein, fat, calcium, and other nutrients. Deactivated yeast biomass may provide additional physical cell shielding to the probiotic organism.

Both trehalose and inulin proved to be better than skim milk as cryoprotectants.

Initial survival offreeze drying Deactivation of microorganisms can occur during centrifuging, lyophilisation and in storage. Centrifugation had a negligible effect on cell viability ofL. rhamnosus LCSH1 and B. lactis BDBB2, as cells showed no difference in calculated to observed concentration after freeze drying, where any cell deactivation through centrifugation would have been apparent Figure 1 d) and From this experiment, it is impossible to determine if losses observed in L. acidophilus MLAJ1 were due to centrifugation or freeze drying or a combination of both.

The best initial survival during freeze drying was afforded by inulin and trehalose, regardless of whether cells were cultured in SPY 2 or SPY 6. Both cryoprotectants produced the largest population of viable and bile tolerant cells.

Acidification activity Acidification activity was not significantly affected by cryoprotectant, although cells freeze dried in trehalose, inulin and skim milk maintained the highest acidification activity per cell.

B. lactis BDBB2 had the greatest acidification activity per cell. It was noted that acidification activity per cell increased during the storage for B. lactis BDBB2 when cultured in SPY 2 (Figure This observation remains unexplained, as cells would not 30/07/02,mcl 221 5 -26have the opportunity to repair cell damage during freeze dried storage and thus increase activity. One possible mechanism is an increase in cell permeability during storage allowing a greater flux of acidic products into the surrounding medium. If present, the change in permeability did not affect cell viability.

Probiotic inhibition of pathogens The pathogen inhibition trials are only indicative of potential probiotic action, as the microflora in the gastrointestinal tract are subject to a range of different exogenous factors from the host, resident and transient bacteria. The agar plates were incubated anaerobically, as conditions in the gut would be anaerobic, reducing the effects of hydrogen peroxide accumulation (Tagg, et al., 1976). The plate technique employed in this research was not buffered (although buffering would occur in the gastrointestinal tract), as preliminary research determined that the strains ofB. lactis did not grow (data not shown) on the buffered media described by Chateau et al. (1993).

E. coli and L. monocytogenes were used for the pathogen inhibition trials. E. coli is naturally present in the gastrointestinal tract and is commonly used as an organism for inhibition trials. Listeria monocytogenes, a Gram positive bacillus, is a cold-tolerant, foodborne pathogen that can cause illness by attacking and multiplying within the gut epithelium. This organism could potentially be inhibited in vivo by a suitable probiotic.

Listeria monocytogenes, has been examined for inhibition by probiotic organisms, along with the observation from this work that Listeria monocytogenes was more sensitive than E. coli, to the effects of the probiotic organisms.

Probiotic organisms freeze dried in inulin and trehalose produced the greatest inhibition ofE. coli and L. monocytogenes.

B. lactis BDBB2 had highest acidification ability per cell and also very high cell concentrations, thus a greater ability to produce acid, but produced the smallest pathogen inhibition out of the organisms tested. Lactobacillus inhibition of pathogens was tested using growth on MRS, whereas B. lactis BDBB2 was tested on RCA, containing only a quarter of the amount of glucose of MRS. L. acidophilus MJLA1 and L. rhamnosus LCSH1 are able to ferment trehalose, whereas B. lactis BDBB2 can not, so the trehalose cryoprotectant can serve as an extra carbohydrate source for those strains. None of the strains are able to utilise inulin as a carbohydrate source, despite inulin being described as a 'bifidogenic' substance (Roberfroid, 1993). Alternatively, pathogen inhibition may not be related to acid production.

30/07/02,mcl 2215.speci,26 27 This method of testing probiotic ability to inhibit pathogens has its own limitations.

The test is sensitive to many exogenous features such as batch of growth medium, incubation temperature and time. Furthermore, there are no defined specifications for inhibition, such as would be used with antibiotic assays, preventing the definitive classification of probiotic organisms as 'inhibitory' or 'non-inhibitory' towards pathogens.

The relevance of a probiotic organism inhibiting E. coli by either 10mm or 14 mm is questionable.

Optimisation of cryoprotectant There were differences in the ability of each cryoprotectant to prevent cell deactivation and freeze injury during freeze drying compared to ongoing survival during prolonged storage. Soy protein, while not protective during freeze drying, was one of the most protective during storage, with cell numbers remaining fairly constant. The biggest changes in cell viability occurred during freeze drying, rather than storage, indicating the emphasis required on adequately preserving cells during the initial freeze drying stage.

Trehalose and inulin were selected for optimisation of initial cell survival, as these cryoprotectants produced the best storage results. The initial deactivation of cell freeze dried in soy protein was so low it could not be considered for optimisation. L. acidophilus MJLA1 was selected as the test organism, as it suffered the biggest decrease in cell viability during freeze drying, compared to the other organisms, thus being the most sensitive to freeze drying.

Optimising the action of cryoprotectants by varying concentration and including antioxidants in the freeze drying medium, identified that the best cell viability and least sublethal injury was achieved with inulin trehalose inulin or trehalose (15%)/ascorbic acid (4 mg/L). Ascorbic acid has been shown to improve survival ofL. acidophilus but not bifidobacteria, in yoghurt produced with mixed cultures. Better survival of freeze drying was achieved using these cryoprotectant solutions, than any of the combinations used in the storage experiment.

The use of either trehalose or inulin as cryoprotectant for probiotic organisms has proven to be better than the reference cryoprotectant skim milk at preventing initial deactivation and sub lethal injury, and maintaining cell viability during storage. Increasing the cryoprotectant concentration to 15% may further improve these results, by increasing the survival of the freeze drying process.

30/0 7 /02,mcl 221 5.speci,27 28 Trehalose (a-D-glucopyranosyl-D-glucopyranose) has been reported to be an effective cryoprotectant for yeasts and bacteria. Trehalose like other disaccharides, is thought to be an effective cryoprotectant due to its ability to form a glass and stabilize phospholipids bilayers in the cell.

Inulin has not been previously reported for use as a cryoprotectant. Trehalose is expensive and not a commonly used food ingredient. Inulin is less expensive and this coupled with other desirable qualities, such as being soluble fibre, fat replacer and 'bifidogenic' factor (Orafti Aandorenstraat 1, 3300 Tienen, Belgium), make inulin an excellent replacement for skim milk.

Conclusions Using the fortified growth medium SPY 6, and inulin or trehalose as cryoprotectants, it is possible to produce freeze dried probiotic cultures, that retain good cell viability and probiotic features both after freeze drying and subsequent storage.

Example 2 A frozen soy dessert can be prepared using freeze dried microorganisms as set forth in Example 1.

Frozen soy dessert All product ingredients (soy beverage, sugar, oil, stabiliser and salt) are combined and heated to 60 0 C for 10 min, then aged at 4°C, overnight. Freeze dried probiotic strains prepared as in Example 1 with Inulin as a cryoprotectant, are individually added (2% inoculum) to the soy dessert base and evenly dispersed by mixing. The product is then churned and frozen (Breville II Gelataio 1600). The frozen soy dessert can then be packed into containers, sealed and hardened to -20°C. The samples are to be stored at -20 0

C.

The product pH is 7.0 0.2.

The cryoprotection ability of inulin in the product can also be increased, by addition of extra inulin to the product base.

Example 3 A yoghurt can be prepared by using starter culture inoculum freeze dried as in Example 1 for the initial fermentation of base ingredients, followed by the optional addition of probiotic cultures freeze dried in inulin.

30/07/02,mcl 2215.speci,28 -29- Yoghurt The base ingredients of milk and milk powder are combined and heated to 85 0 C for and tempered to 43 0 C. The base is then inoculated with starter cultures, Streptococcus thermophilus and Lactobacillus bulgaricus freeze dried in inulin as in Example 1, at a level of 106 cfu/mL. The yoghurt base is then incubated at 43 0 C until a pH of 4.5 has been achieved. Freeze dried probiotic microorganisms, such as L. acidophilus MJLA1 freeze dried in inulin, are added to the mix at a concentration of 106 cfu/mL and evenly dispersed. The base is then dispensed into plastic tubs, sealed and stored at 4 0

C.

Extra inulin (up to can be included in the yoghurt base to aid cryoprotection of probiotic microorganisms during storage at low temperatures.

Industrial Applicability The present invention has application in the food industry, with respect to the preparation of fermented and probiotic foods and health supplements.

30/07/02,mcl2215.speci,29 References Bernet, M. Brassart, Neeser, J. R. and Servin, A. L. (1993) Adhesion of human bifidobacterial strains to cultures human intestinal epithelial cells and inhibition of enteropathogen cell interaction. Applied and Environmental Microbiology 59: 4121-4128.

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Chateau, Castellanos, I. and Deschamps, A. M. (1993) Distribution of pathogen inhibition in Lactobacillus isolates of a commercial consortium. Journal of Applied Bacteriology 74: 36-40.

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30/0 7 /02,mcl 2215 speci,31 -32- Rybka, S. and Fleet, G. H. (1997) Populations ofLactobacillus delbrueckii ssp bulgaricus, Streptococcus thermophilus, Lactobacillus acidophilus and Bifidobacterium species in Australian yoghurts. Food Australia 49: 471-475.

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30/07/02,mcl 2215.speci,32

Claims (37)

1. The use of an oligo/polysaccharide comprising branched glucose and fructose chains of heterogeneous lengths as a cryoprotectant for microorganisms.

2. The use according to claim 1 wherein the oligo/polysaccharide is of plant origin.

3. The use according to claim 2 wherein the plant is selected from the group consisting of topinambour, chicory, onion, asparagus and artichoke.

4. A method for freeze drying a microorganism, which method comprises freeze drying the microorganism in the presence of an oligo/polysaccharide comprising branched glucose and fructose chains of heterogeneous lengths. A method for preventing cell deactivation during freeze drying of a microorganism which method comprises freeze drying the microorganism in the presence of an oligo/polysaccharide comprising branched glucose and fructose chains of heterogeneous lengths.

6. A method for preventing sublethal injury during freeze drying of a microorganism, which method comprises freeze drying the microorganism in the presence of an oligo/polysaccharide comprising branched glucose and fructose chains of heterogeneous lengths.

7. A method for enhancing storage survival of a microorganism which method comprises freeze drying the microorganism in the presence of an oligo/polysaccharide comprising branched glucose and fructose chains of heterogeneous lengths.

8. A method for freezing a microorganism, which method comprises freezing the microorganism in the presence of an oligo/polysaccharide comprising branched glucose and fructose chains of heterogeneous lengths.

9. A method for preventing cell deactivation during freezing of a microorganism which method comprises freezing the microorganism in the presence of an oligo/polysaccharide comprising branched glucose and fructose chains of heterogeneous lengths. A method for preventing sublethal injury during freezing of a microorganism, which method comprises freezing the microorganism in the presence of an oligo/polysaccharide comprising branched glucose and fructose chains of heterogeneous lengths. 30/07/02,mcl 2215.speci,33 If -34-

11. A method for enhancing storage survival of a microorganism which method comprises freezing the microorganism in the presence of an oligo/polysaccharide comprising branched glucose and fructose chains of heterogeneous lengths.

12. A method for enhancing storage survival of a microorganism which method comprises storing the microorganism in the presence of an oligo/polysaccharide comprising branched glucose and fructose chains of heterogeneous lengths.

13. The method according to any one of claims 4 to 12 wherein the oligo/polysaccharide is of plant origin.

14. The method according to claim 13 wherein the plant is selected from the group consisting oftopinambour, chicory, onion, asparagus and artichoke. The use of inulin as a cryoprotectant for microorganisms.

16. A method for freeze drying a microorganism, which method comprises freeze drying the microorganism in the presence of inulin.

17. A method for preventing cell deactivation during freeze drying of a microorganism which method comprises freeze drying the microorganism in the presence of inulin.

18. A method for preventing sublethal injury during freeze drying of a microorganism, which method comprises freeze drying the microorganism in the presence of inulin.

19. A method for enhancing storage survival of a microorganism which method comprises freeze drying the microorganism in the presence of inulin. A method for freezing a microorganism, which method comprises freezing the microorganism in the presence of inulin.

21. A method for preventing cell deactivation during freezing of a microorganism which method comprises freezing the microorganism in the presence of inulin.

22. A method for preventing sublethal injury during freezing of a microorganism, which method comprises freezing the microorganism in the presence of inulin.

23. A method for enhancing storage survival of a microorganism, which method comprises freezing the microorganism in the presence of inulin.

24. A method for enhancing storage survival of a microorganism which method comprises storing the microorganism in the presence of inulin. 30/07/02,mcl 2215.speci,34 A culture of a microorganism which has been prepared using one or more methods according to any one of claims 4 to 14 or 16 to 24.

26. A microorganism according to claim 25, wherein the microorganism is selected from a microorganism used in the preparation of a food and a probiotic microorganism.

27. A food incorporating one or more microorganisms according to claim 26 prepared by a method of any one of claims 4 to 14 or 16 to 24.

28. A food prepared using one or more microorganisms according to claim 26 prepared by a method according to any one of claims 4 to 14 or 16 to 24.

29. A food according to claim 27 or 28, wherein the food is a cold food. A food according to claim 27 or 28, wherein the food is a frozen food.

31. A food according to any one of claims 27 to 30 wherein the food is vegetarian food.

32. A food according to any one of claims 27 or 29 to 31 wherein the food is prepared by adding the one or more microorganisms to the already prepared food.

33. A food according to any one of claims 27 or 29 to 31 wherein, the one or more microorganisms is added to the food during preparation.

34. A food according to claim 28 wherein the food is prepared using one or more microorganisms and then has further microorganisms added.

35. A microorganism according to claim 26 prepared in capsular form with an oligo/polysaccharide comprising branched glucose and fructose chains of heterogeneous lengths present in the encapsulated form.

36. A microorganism according to claim 26 in tablet form with an oligo/polysaccharide comprising branched glucose and fructose chains of heterogeneous lengths present in the tablet.

37. A microorganism according to claim 26 prepared in capsular form with inulin present in the encapsulated form.

38. A microorganism according to claim 26 in tablet form with inulin present in the tablet.

39. Use of a microorganism according to any one of claims 35 to 38 as a health supplement. Use of a microorganism according to any one of claims 35 to 38 for incorporation into a food. 30/07/02,mcl 2215.speci,35 0 -36-

41. A method for extending the shelf life of a food containing one or more microorganisms which method comprises incorporating an oligo/polysaccharide comprising branched glucose and fructose chains of heterogeneous lengths in the food.

42. A method for extending the shelf life of a food containing one or more microorganisms which method comprises incorporating inulin in the food.

43. A method for increasing the survival of one or more microorganisms in a food or health supplement which method comprises incorporating in the food or health supplement an oligo/polysaccharide comprising branched glucose and fructose chains of heterogeneous lengths.

44. A method for increasing the survival of one or more microorganisms in a food or health supplement which method comprises incorporating inulin in the food or health supplement. DATED this 3 0 th day of July, 2002 THE UNIVERSITY OF NEWCASTLE RESEARCH ASSOCIATES (TUNRA) LTD. By their Patent Attorneys: CALLINAN LAWRIE 30/07/02,mcl 2215.speci,36