CA2420095A1 - Cryoprotection - Google Patents

Abstract

The invention relates to methods of protecting microorganisms against lethal and sub-lethal damage caused by exposure to low temperatures. Protection involves using oligo/polysaccharides.

Description

_. _2_ CRYUPROTECTIQN
'technical Field This invention relates to the protection of microorganisms against lethal and sub-lethal damage caused by exposure to low temperatures. This protection is of use in froxen microorganisms and freeze drying microorganisms and in the preparation and storage of fermented and probiotic foods.
>sackgrpund Art ro 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 ingredieztts; or ii) probiotic microorganisms can be added in suitable concentrations to the finished product prior to packaging (l3ulIimore; 1983; Gilliand, 1985). Starter and probiotic cultures can be 15 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.
Froaen 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 2o 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 tactic acid bacteria cultures are still in an experimental stage and 25 good cell viability is not yet standard (To and Etzel, 1997)_ Freezing and freeze drying microorganisms can affect their viability.
Gryoprotectants 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 3o maximum viability, metabolic activity pr 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, gums, maltodextrin; and antiaxid$iits: 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, x0 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.
Aiirer freeze drying, probiatic cultures should preferably retain both ma~cimum viability and activity, or the specific attributes associated with that isolate.
For probiotic microorganisms to be of benefit to the consumer, they must be 1s present iz~ the food or tablets in a suitable concentration. When incorporated into a product, probiotics need to remain viable at a concentration of lOdcfulgram or greater for the entire shelf life of the product to be of benefit to the consumer. A
person vrould then need to consume at least 100 grams of the product every day to ensure a minimum daily , dose of probiotics of l O8cfu in total, in order to gain health benefits.
20 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 frrmented dairy 25 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 attd bile conditions encountered in the ,gastrointestinal tract. Product manufacture may injure probiotic cells. Although they remain viable, impaired cells are readily inactivated if less 3o than ideal conditions are experienced, for exarmple, 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 SS 13-galactosidase activity or bile tolerance.

- !~ -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 a-D~-G1u-( 1-2)-[(~-zo D-Pru-(1~2r~° (Crittenden, 1999). Inulin, marketed as RaRiline {Orafti, Aandorenstraat 1, 3300 Tienen, Belgium), is non-digestible to humans, but acts as a 'prebiotic', selectively being utilised by Biftdobacterium species in the human gut (Roberfroid, 1993).
The information supplied by the marketing company reports that the strain B, lactic Bb-12 can utilise inulin although the test conditions 'are not mentioned. Inulin has a low calorific 15 content and also acts as soluble dietary fibre. Inulin is already used in foods intended as dietary aids, with fibre, fat repla,cer and improved te~ctural qualities.
The present inventors included the prebiotac inuiin 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 2o 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 aligolpolysaccharide comprising branched 25 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 oligolpolysaecharide include topinambour, chicory, union, asparagus and artichoke.
In particular the present invention provides use of inulin as a cryoprotectant for 3o microorganisms.
The present invention provides a method for freeze drying a microorganism, which method comprises freeze drying the microorganism in the presence of an oligolpolysaccharide comprising branched glucose and fructose chains of heterogeneous lengths.

The present invention provides a method fox freeze drying a microorganism, which method comprises freeze drying the microorganism in the presence of inulin.
The present invention provides a method fQr 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 preverning cell deactivation during freeze drying of a microorganism which method comprises freeze drying the microorganism in to the presence of an oligolpolysaccharide 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.
15 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 oligolpolysaccharide comprising branched glucose and fructose chains of heterogeneous lengths, The invention provides a method for preventing cell deactivation during freezing of 2o a microorganism which method comprises freezing the microorganism in tlae 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 oligolpolysaccharide comprising branched glucose and fructose chains 25 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 30 a microorganism, which method comprises freezing the microorganisrtt in the presence of an oligolpolysaccharide comprising branched glucose and fructose chains of heterogeneous lengths.

.. _ The invemion provides a method for preventing sublethal injury during,freezing of a microorganisnn, which method comprises freezing the microorganism in the presence of inulin.
The invention provides a method for enhancing stoiage survival of a microorganism which method comprises freeze dt3ring the microorganism in the presence of an oligolpolysaccharide comprising branched glucose and fructose chains of heterogeneous lengths, The invention provides a method fox enhancing storage survival of a microorganism which method comprises freeze drying the microorganism in the presence to 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.
15 The invention provides a method for enhancing storage survival of a microorganism which method comprises $eezing 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 zo oligoJpolysaecharide 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 presexice of inulin.
25 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 3o microorganisms prepared by a method of the inve»tion.
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 ofthe 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°C.
In another aspect the present invention provides a frozen food incorporating one or more microorganisms prepared by a method ofthe invention. The frozen food is a food which is maintained at a temperature below its freezing point. Typically the storage temperature is about -20°C.
rn 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 mom temperature but above freezing.
Typically the storage temperature is about 4°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 ~20°C.
13 In yet another aspect the present invention provides a vegetarian food incorporating one or more zx~icroorganisms 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 invernion. The vegetarian 2o 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 microorganisms) within the food and 25 resultant effects on the properties of the food. The microorganisms) may participate in fermentation of raw materials in the preparation. The microorganisms) may provide partial or complete fermentation ofraw materials in the preparation. The microorganisms used in this way may only provide fermentative functions or may provide fermentation and probiotic functions.
3o 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 .. g _ oligolpolysaccharide 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 microorisms 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.
14 The microorganisms) may be prepared in capsular form with the oligolpolysaccharide comprising branched glucose and fructose chains of heterogeneous lengths present in the encapsulated form. The microorganism{s) in this farm may be used as a health supplement or may in turn be incorporated into a food.
The microorganisms) may also be prepared in tablet form with the ~5 oligolpolysaccharide 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 ZO inulin in the food or supplement.
The microorganisms) may be prepared in capsular form with inulin present in the encapsulated form. The microorganisms) in this form may be used as a health supplement or may in turn be incorporated into a food. The microorganisms) may be prepared in tablet form with inulin present in the tablet. The microorganism{s) in this form ttaay be used as a zs health supplement or incorporated lento 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 3o probiotic microorganisms ensures they will survive the passage through the gastrointestinal tract, and as long as they are present in su~cient numbers, will confer their particular health benefits to the consu~r.
lactic acid bacteria and probiotic cultures for commercial food production, and starter cultures for fermentation can be purchased from international companies such as _g_ Christian Hansen Fty Ltd (Bayswater, Australia) and Gist-Brocades Australia Pty Ltd (Mooreban~, Australia). Other research organisations such as the CST)~O
Starter Culture Collection (Highett, Australia) and the Australian Starter Cuhure Research Centre (Vflerribee, Australia), have goad lactic acid bacteria and potential probiotic collections but these organisms are only available on a small scale.
Fable 1 Species currently used as probiotics around the world.
Lactobacillus spp. ~~~obact~~m spp.

L. acidophilus $. biftdum L.,~ohnsonii .8, lon~m L. paracasei ssp. Paracasei .~, infantis L. rhamnosus ,8. brev~

L. plantarum B, adolescentis L. brevis B. lactic L. reuteri L: salivarius L. fermetum L. helveticus L. delbrueckii ssp.
Bulgaricus Uther Species Streptococcus salivarius ssp_ Thermophilus .Laciococeus lactis ssp. Lactic and cremoris Enterococcus faeciurn Leuconostoc mesenteroides ssp. l7extranium Propionibacterium, freudenreichii Pediococcus acidflactici Saccharomyces boulardii Escherichia colt Bacteroides spp.
Bacillus spp.
Adapted from O'Sullivan et al. (1992), Sanders (1999) and R.olfe (2000) Selection criteria for prohiatic 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 , 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 o~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 goad 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 cori~rzr~ed 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 identi~ ed ~ The strain must be clinically safe far 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 gastroimestinal 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 Adapted ~rom Klaenhammer and Kullen (I999) and Cribson and Fuller {2000) Host origin Frobiotic microorganisms should be of human origin if they are intended for human consumption. Not only is this a safety consideration in terms of crass-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 to in which they are intended to be used .
Safety Safety in consuming live cultwes is one of the most important factors when examining potential probiatic microorganisms. Lactobacillus (with the exception ofL.
rhamnosur), Bifddbaeterium and S baulardif 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. Detertnir~e the intrinsic properties of the strain - antibiotic resistance, plasmid transfers etc. ,

2. Assess tlae 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 1~ -Taken from Donohue and Salminen ( I 99~
,Survival in thegastrointestinal tract 'USl'hen 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 Lactabacillacs species preferably colonise the ileum. For best survival rates, strains need to be acid, bile and lysozyme tolerant to provide a competitive advantage in viva, Bile and acid resistance in probiotic rniraroorganisms has been trialed in vitro, using batch and ZO multiple chemostat techniques, with media containing appropriate levels of bile or buffered at low pH to mimic the gastrointestinal system. In vitro experimems are not ideal, but do effectively highlight inadequate species and provide a staxti~ng point for further experimentation.
Intes~irtal adhesion or colonisation .
1s Ideally, it is desirable that probiotic nnicroorganisms adhere to or colonise the intestine. The benefrts of colonisation include displacement or e~cclusion of undesirable bacteria or pathogens from adhering to the intestines and prolonged existence of desirable bacteria in the intestines. The longer probiatic microorganisms are present in the intestines, the greater any possibility of beneficial effect on the host.
2o Although coionisatian does not appear to be permanent, delayed residence in the large intestine is apparent ~uvith 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 25 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. (1893) showed that several species of B~do6acterium could inhibit cell adhesion and invasion by E. coli and S typhimurium. Other similar in vitro cell culture 3o experiments have been conducted, all reporting strain specific adhesion, put of 12 Lactabacidlus strains only 4 strains, L. casei 744, L. acidophilus Lal, l..
rhamnosus r.C-7~5 and L. rhamnasus CxCx, displayed significantly greater adherence than the non specific binding of E. coli.

Industrial exploitation For industrial food production, a probiotic strain must be technologically exploitable. rt 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.
to Surwfval 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.
delbrrreclaii ssp. ~bulgaricus and Streptococcus therrnophilus do not survive in the gastrointestinal tract environment, but they do have excellent industrial properties. Good 15 manufacturing and survival characteristics are not inherent in all lactic acid bacteria, indicating that strain selection is az~ irnpQrtant 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 1 O8 - 109 2o cfu per day is a commonly quoted figure for adequate probiotic consumption, equating to 1008 of a food product containing 106 - 10' cfu/g.
'>rhe form in which probiotic bacteria are fed, affects the minimum dose for detection in the faeces. L. rhamnosus CrCr could be detected in host faeces when fed at a lower concentration of I0~ cfu in fermented or sweet milk and as enterocapsules, rather zs than at 10'°cfu in a freeze dried capsules. "fhe difference in survival was attributed to the milk buffering capacity and insoluble enterocapsute 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 o (Standard ~8) stipulates that yoghurt must have a pl:I less than 4.5 and be prepared with S
thefmophilus and 1,. delbrueckii ssp. bulgaricus or other suitable cultures, but does not stipulate required levels of probiotic bacteria. Some countries have imposed loose standards o~ minimum allowable levels of probiotics or lactic acid bacteria in yoghurts.

-. -14-There appear to be no specifications with regard to prabiotic products that are not of dairy origin.
Probiotic survival in products is affected by a range of factors including pH, post acidif ration, hydrogen peroxide production, storage temperature, the mixture of starter cultures, packaging and food ingredients. Bii~xdobacteria 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 cap~ule~
1o Tablets and capsules are prepared in accordance with standard technidues 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 treat prior to use. The present invention relates to use of inulin in this form 15 but also relates to use of other forms of inulin whether heat sterilised or not. Similarly, the present invernion relates to use of an oligolpolysaccharide comprising branched glucose and fructose chains oFheterogeneous lengths as a high purity gel which is sterilised with heat prior to use. The present invention also relates to use of other forms ofthe oligoJpolysaccharide whether heat sterilised or not, The desirability of sterilising materials 20 for use in growing or storing microorganisms will be sel:Fevident to the skilled addressee, as will be the fact that sterilisation can be carried out in other ways.
D~nitions Oligosaccharide: a glycoside containing between three and ten sugar moieties Polysaccharide: a glycoside containing between three and eight' sugar rrtoieties 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 mare other features, integers, steps, components or groups thereof.
Cryoprotectant: a cryoprotectant is any compound that protects biological material or cells from the detrimental e~'ects of cold temperatures in preparation or storage.

. r _ 1fJ _ Brief Description ofthe Drawings Figure 1 shows the expected and observed cell concentrations after freeze drying in various cryoprotectants, for probiotic bacteria grown in SPY 2 anr! SPX S.L.
acidophilus MrLAl grown in a) SPY 2, b) SPX 6; .1.. rhamnosus LCSH1 grown in c) SPY 2, d) SPY 6; and B. lactic 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 cryopratectants: a) L.
acidophilus MJLA1, b), L. rhamnorus LCSH1 and c) B. lactic BDBB2 grown in SPY 2 (solid circles) and SPY 6 (solid triaxlgies) when recovered on agar (open symbols) and agar + 0.3% bile (closed syrnbols)_ Figure 3 shows the inhibition by L. acidophilus Ml"LA~ of E, coli after growth in a) SPY 2, or b) SPY 6, and L. monocyto~enes after growth in c) SPY 2, d) SPY 6 and freeze drying in various cryoprotectants.
is Figure 4 shows the inhibition by ~,, rhamnosus LCSI~1 of E. coli offer growth in a) SPY 2, or b) SPY 6, and ,~. monocytogenes aRer growth in c) SPY 2, d) SPY 6 and freeze drying in various cryoprotectants.
Figure 5 shows the inhibition by $. lactis BDBB2 of E. coli after growth in a) SPY 2, or b) SPY 6, and L. monocytogenes after growth in c) SPX 2, d) SPY 6 and freeze drying 2o in various cryoprotectants.
Figure 6 shows the acidification activity of probiotic organisms after freeze drying in various cryoprotectants: L. acidophidus MJLAI grown in a) SPY 2, b) SPY 6; L.
rhamnasus LCSH1 grown in c) SPY 2, d) SPY' 6; and B. lactic BDBB2 grown in e) SPY 2, f) SPY 6 and freeze dried in cryoprotectants.
Best Method of Carrying Oat 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 e~tcellertt 3o cell viability and retention of probiotic characteristics. The cryoprotectant acts as a replacement for non-fat skim milk (NFSM), the most commonly used cryoprotectant. The use ofNFSM as a cryoprotectant makes subsequent use ofthe microorganisms unsuitable for consumption by vegetarians ox people with milk allergy ar hypersensitivity.
According to the literature, the ideal cryoproteeta.nt needs to bind water, prevent ice crystal formation, protect cell membranes, enhance cell shielding, prevent oxidation and eradicate free radicals. No realistic replacement far skim milk as a cryaprotectant has been identified prior tv the present invention.
Exogenous conditions that affect suz-vival of freeze drying include method and time of cell harvest and correct storage of freeze dried powders. Cells should be harvested at 1o 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 frltrataon or ultrafiltratian rather than centrifugation, however, better survival occurred in cells obtained by centrifugation or ultrafiltration. Turing centrifuging, a higher temperature aids cell separation, but temperatures around 5°C is less detrimental to cell 15 viability.
Bozoglu et al. (19$7) modelled the survival l~inetics of lactic acid bacteria aztd concluded that cell death is related to the area exposed to tha 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 20 enough to be beneficial without causing osmotic problems, approximately 1 Or°cfu/rnL.
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 rwith over drying and under drying both detrimental to cell viability. ')"he dried cultures should be kept under vacuum or nitrogen gas, but not air or oxygen gas, to prevent oxidation. The 2s cells retain greater viability when stored at refrigerated temperatures of

5°C and below-The packaging should be moisture proof, oxygen proof and opaque.
ll~aterials and R~ethods Microorganisms 30 Probiotic cultures Lactobacillus aciclo,~lailus MJLAI, Bi~dobacterium lacdis B)(7BB2, and Lactobacillus rhamnosus LCSHI, were supplied by Christian Hansen (Bayswater, Victoria Australia) and used in freeze drying cryoprotectant experiments. L.
acidophilus MJLAl was used in the cryoprotectant concentration and antioxidant trials.
NX'icl'obiological growl)t 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 7,4 using HCl or NaOT~, the medium dispensed into bottles and autoclaved at 121 °C
for 15 rains. 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°C for 15 rains and then further 1o supplemented ~avith sterile CaC12.2HZO and MnC12.4H~0, to a. final concentration of 6.0 mM each, MRS medium: de Man, Rogosa and Sharpe medium (de Man et al., 1960) RCM medium: Reinforced Clostridial medium (Hirsch and Grinsted, 1954) is TSA medium: Tryptone Soya Agar (Oxoid Australia) NB medium: Nutrient Broth (Oxoid Australia) PBS: Phosphate buffered saline, (lMaC18.OOg, KCl 0.208, Na2HPO4 1.44g, KlrI2P0° 0.248, distilled water 1 litre, pT~ 2.0) zo Example 1 Freeze Drying Preservation of Cultures Probiotic cell production and han~est Bacteria were grown in of SPY 2 and SPY b (I Litre) for 24 hours at 37°C.
Lactobacilli were incubated in 8% COz and bifidobacteria incubated in aerobic 2s atmospheres. Cells were harvested by centrifugation (7000 x g at 4°C, 6min) and resuspended in each cryoprotectant (SOmL) (Table 5), to give an approximate concentration of 10'° cfulmL. 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°C). Freeze 30 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 -stored in the dark, under vacuum (-0.6 mbar) at S°C. F,ach experiment was conducted in triplicate.
Table 5 Compounds used as cryoproteetants for preserving various probiotic organisms daring freeze-drying.
Cryoprotectant Sterilisation treatment Noz~-fat skim milk (Diploma) 10% pH 6.6, inspissated 100°C, l5mins Inulin HP-Gel ((~rafti) 10% pH 6.3, inspissated 100°C, l5mins Yeast biomass (Sigma) 10% pH 7.0, inspissated 100°C, l5mins ?rehalose (Sigrma) 10% pH 7.0, autoclaved 121°C, l5mins Soy milk pH 7.0 (+/- 0.2), ZJH~T
(Sanitarium Health Food Co, Australia) commercial package Soy protein isolate 5% pH 7.0, micro~uidixed at 7500 PSI, (IPT 545, International Protein Technologies) inspissated 100°C, l5mins All cryoprotectant solutions were suspended in distilled water and sterilised as stated above_ All cryoprotectants were used for microorganisms grown in $PY2, and skim milk, trehalose, inulin and yeast biomass were used for microorganisms grown in SPY 6.
x0 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 (Ioomg, 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 !5 RCA agar and ItCA + 0.3% bile (Oxoid) far bifidobacteria. Plates were incubated for 48 h at 37°C, lactobacilli at $% CO2 and bifidobacteria incubated anaerobicaily, after which the resulting colonies were counted. Cell concentrations were calculated as cfulg powder.
Survival o~ freeze dtyi~ag 24 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 ofgrowth medium 2s (cfu), divided by the final weight of the freeze dried powder (g). the total colony forming units in 1 titre of SPY 2 or SPY b medium was calculated using maximum population data for each organism from previous growth medium trials, Pathogen inhibition Bacteria were assessed far ability to inhibit .E'. coli (NCTC 11560) and Listeria monocytogerter (ATCC 7644) in vitro based on the methods of Chateau et al.
(1993). Two aliquots {10 p.I,) of rehydrated cell suspension was spotted onto two MRS or RCA agar plates and incubated for 24h anaerobically to prevent H20z accumulation, Plates were then overlayed with Tryptone Soya Agar (TSA; Oxoid) containing 0.1 mL of an overnight 1o Nutrient Broth (Oxoid) culture of either E. coli or L. rrrortocytogenes.
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.
Acid~cation activity Each rehydrated cell slurry (0.2 rnl,) was inoculated into soy milk (2mL) (So Good, Sanitarium Health food Co, Australia) that had been ternpexed to 37°C.
The pH of the uninoculated and inoculated soy milk (pH;~;~;,, ) was measured, The inoculated soy milk samples were then incubated at 37°C for a hours, the lactobacilli in 8°!o COz and bifidobacteria anaerobically, after which the pH (pH~;,r"~) was measured. CeII
activity vcras calculated as change in pH per hour per log cfulmL, using the following equation:
Cell Activity = dpH (pH;~;~ - pH r",u) time {hours) x (Log,QCfu/mL) Cryoprotectanr concentration and antioxidants L. acidaphilus strain MTLA1 was inoculated into SPY 6 medium (IOmL) and incubated far 20 hours at 37°C in 8% COa,. Cells were then harvested by centrifuging (5000 x g, 5 mitt), the supernatant discarded and the pellet resuspended in each 3o cryoprotectant (I.OmL}. Cryoproteetants used in this experiment are listed in An aliquot (O.8mL} of the resuspended cell concentrate was dispensed into an eppendorf tube, frozen to -80°C and then freeze dried overnight (-40°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 ~Za-chloride solution for 24 hours.
Immediately after water activity equilibration, each tube was rehydrated to initial volume in viability and activity for immediate use tests. Each cryopmtectant was trialed in Table

6.

Table 6 Cryoprotectant suspension solutions for freeze~drying L.
acidophilus strain 11~JLA1.

Cryoprotectant Treatment Control distilled Hz0 pH 7.0, autoclaved i21C, l5mins ! Trehalose~(S%).._______________.__._................_.__.___~H 7.0, autoclaved 121C, l5mins Trehalose (10%) Trehalose (15%) ,..... ..___.__..__.__ -.
_ .....
.
.

__ ~H 6.3, inspissated 100C, _________.__ l5mins _ .
.
Inulin~(5%) _ Inulin (10%) , Inulin ( I S %) .
.

. .pH 6.7, inspissated .... 100C, l5mins ..Trehalose (2.5%) +~inulin (2.5%) y___________..

Trehalose (5.0%) + inulin {5.t~%) Trehalose (7.5f ) + inulin (7.5%) Trehalose (15%) + tacopheral~(Sigma)~(IOULIL)_pH 7.0, trehalose solution'___________ _-_ Trehalose (15/a) + tocopheral (100u~)autoclaved 121C, XSz~nins.
Sterile ~'rehalose (15%) + ascorbic acid antioxidant added when (Sigma) (4mgJL) cool.

Trehalose (15%) + ascorbic acid (40mglL,) _ pH 6.3, inulin solution Inulin (1 S%) + tocopheral {10pL%L)inspissated ~ ~ ~

InuIin {15%) + tocopheral (100uLIL)100C, l5mins_ Sterile antioxidant Inulin (15%) ~- ascorbic acid (4mgli,)added when cool.

Inulin (15%) + ascorbic acid (40mg~i.) CeII 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 IvIR,S + 0.3°!o 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 {O.1mL) was mixed with phosphate buffered saline (1.0 mL, PBS, pH 2.0). 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.

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 (p<0.05). Trehalose was the next best, 15. followed by skim milk and then yeast biomass. There were no significant di:El:'erences betvuee~ cryoprotectants with respect to sub-lethal injury of cells.
Survival of freeze 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 2o the decline in population. The angles were compared using AN4VA. The rate of cell deactivation during storage depended ari the growth medium, eryoprotectant and the bacterium (p~0.05) When probiotic cells were cultured izt S>fY 2 (Figure 2), the cryoprotectants that provided the best storage protection were trehalose and soy protein. Cell viability in these 25 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 3o tolerance. Yeast biomass was not as e~'ective in protecting cells (p~0.05).

. . -22-Probiotic Xnhibition 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 ofprobiotic 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 {p<0.05). z. acidophilus MJLAI {figure 3) and ~. rhamnosus LCSI31 (Figure 4) gave greater inhibition when they were grown in SPY 6 prior to freeze drying.
Inhibition by B.
to lactrs BDBB2 (Figure 5) was not affected by growth medium.
fl cidiftcation activity The acidification activity per cell of freeze dried probiatic organisms during storage is presented in 'Figure 6.
When probiotic organisms were gror~m in SPY 2, using multi-factor ANOVA for 15 data analysis, trehalose and inuiin maintained the best acidification activity during storage (p<0.05). Skim milk and soy milk were then next best, followed by soy protein, then yeast bioxnass. Probiotic orgaztisms cultured in SPY 6 prior to freeze drying, retained the highest acidification activity in trehalose and skim milk (p<0.05). Cells stored using yeast biomass as the cryoprotectant, had the biggest decrease in acidification ability during storage.
20 Overall, the decline in acidification activity o~probiotic organisms grown in SPY Z
was lower than that of the organisms grown in SPY 6 (p<0.05). I~owever, there was na significant difference between cryoprotectants, using the results from both growth media.
The variations between bacteria and between media were too great.
G~ryoprotectar~t concentration zs The e~'ect of cryoprotectant concentration and the presence of antioxidants an 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 freeae drying (Table 7). There were differences in acid tolerance prior to freeze drying, depending on the cryoprotectant (p<0.05). The acid tolerance treatment did decrease cell 3U viability prior to and after freeze drying (p<0.01) (Table 8).

Freeze drying caused a significant decrease in cell viability, bite tolerance and acid tolerance (p<0.05). Water, the control, produced the lowest cell viability, bile and acid tolerance after freeze drying (p<0.05)_ Inulin {15%), trehalose (IS%), trehalose (7.5%)linulin (7.5%) 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 I5%
inulin and 15% trehalose. The higher levels of antioxidants were generally detrimental to cell viability.
IO
Table 7 Cell viability and bile tolerance of l,. acidophilus MJLAI after freeze drying in various concentrations of cryoprotectants and antioxidants.
Cell viability Bile tolerance after after freeze freeze drying drying Ctyoprotectant (log cfulml,) (log cfulmL) mean s.d. mean s.d.

Water b.BI ~ 0.1 5.79 ' t 0.25 I

Ttehalose (5%) 8.03' ~ 0.057.61br ~ 0.05 Trehalose (10%) 8,20"'6' t 0.058.07'6 ~ 0.31 Treha3ose (15%) 8.30' ~ 0.048.03"~d t 0.10 Inulun (5%) 8.12 ~ 0.157.67r~' ~ 0.19 Inulin (10%) 8.18'6' ~ 0.067.876~'F t 0.08 Inulin (15%) 8.29' t 0.098.138 t 0.04 Trehalose (2.5%) + inulin 7.89' ~ 0.067.57h t 0.06 (2.5%) Trehalose (5.0%) + inula~a 8.044 t 0.027.81'rB t O.OI
(5.0%) Trehalose {7.5%) + inuliuu 8.25'x' t 0.088.04" ~ 0.
(7.5%) I 1 Trehalose ( I S %) + tocopheral8.21 b' ~ 0.027.90' ~ 0.
( 10~1.,I~.) 03 Trehalose (15%) + tocophera!8.16' t 0.057.83d'f t 0.1 (IOOULIr,) I

Trehalose (I5%) + ascorbic 8.24'6 f 0.057.93"t'cae~ 0.05 acid (4mgIL) Trehalose (15%) + ascorbic $,16~ t 0.037.87~f ~ 0_07 acid (40mgIL) Inulin (15%) + tocopheral 8.10 ~ 0.037.$4d'f t 0_ (10~,LIL) 10 hnulin (15%) + tocopheraf 8.166' ~ 0.047.90a'd' f 0.02 (100pL/L) Inulin (15%) + ascorbio 8.20" f 0.127.95'''' t 0.
acid (4mglL) I 1 InuIin (15%) + ascorbic 8. I4~ t 0.027.85'r t 0.07 acid (40mgJL) s.d. = standard deVildti0ri (ri=3),'' °' e'° significantly different survive! compared to other Cl~'O~rOteC~ntS
IS

_ . -24-Combinations of trehalose and inulin were only successful at the highest concentration that was trialed. Trehalose (5%)linulin {5%) and trehalose (2.5%)rnuliti {a.5%) were not effective at maintaining cell viability, bile tolerance, or acid tolerance at reasonable levels.
')fable $ Acid tolerant cell populations before and after freeze drying in various concentrations of cryoprotectants and antioxidants.
Acid tolerance Acid tolerance after before freeze freeze drying drying Ctyoprotectant (log cfuJmL) (log cfuJmL) mean s.d. Mean s.d Water 8.81' ~ 0.056.278 ~ 0.08 Trehalose (5%) 8.71 t 0.047.87 ~ 0.20 Trehalose (l0%) 8 68' f 0.078.09 f 0.12 Trehalose (15%) 8.73'x' t 0.028_30 ~ 0.08 Inulin (5%) 8.76e'a' f 0.038.07 ~ 0.17 Inulin (I0%) ~ 8.79~ ~ 0.128.09 t 0.11 Inulin (15%) 8.80a~ ~ 0,058.22ab t 0.05 Trehalose (2.5%) + inulin 8.80as' t 0.067.72r ~ 0.12 (2.5%) Trehalose (5.0%} + inulin 8.80"~ ~ 0.017.96 ~ 0,02 (5.0%) Trehalose (7.5%) + inulin 8.$58b ~ 0.058.16~b' t 0,05 (7.5%) ?rehalose (15%) + wcophetal8:74'~e t 0.018.04 t 0.05 (iO~i,Ji,) Trehalose (15%) +tacopheral8.79a~ ~ 0.028.04 ~ 0:03 (100uLIL) Trehalose (15%) + ascorbic 8.85' f 0.036.14'~b ~ 0,09 acid (4mgIL) Trehalose (15%} + ascorbic 8.'788 t 0.038.03' ~ 0.08 acid (~OmgIL) Inulin (15%) -r tocopheral 8:67' t 0.078.04" f 0.02 (lOpLIL) Inulin (15%) + tocopheral 8.75'd' ~ A.Ob8.03"'' t 0.03 (100~LJL) lnulin (15%) + ascorbic 8.78 ~ 0:078.12 t 0.15 acid (4mgl~,) Inu(in (15%) + ascorbic $.73'd' ~ 0.078.04" ~ 0.02 acid (40mg1)_.) s.d. ~= standard deviation (n=3), '' °' "' significantly differenk 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 a~zd 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.
~5 Adding Tween 80 and calcium to growth medium bas been observed to improve suz'vival, without preventing sub-lethal injury. frobiotic bacteria cultured in SPY ~ did retain a higher degree of acidification ability during storage.
Freeze drying cryoproteetnnts As skim milk is the most commonly used eryopratectant for freeze drying bacteria, it was used as a reference cryoprotectant, to which other cryoprotectants were compared in this experiment. The compounds trialed as eryoprotectants 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 to 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.
Initit~l survival o, f~'reeze drying Deactivation of microorganisms can occur during centrifuging, lyophilisation and in storage. Centrifugation had a negligible effect on cell viability of.~.
rharnnosus LCSHl and B. lactic 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 f~. p'rom 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 anulxn az~d trehalose, regardless of whether cells were cultured in SPY 2 or SPY 6. Both cryoprotectants produced the largest population ofviable and bile tolerant cells.
Acidtficatian activity Acidification activity was not significantly affected by cryaprotectant, although cells freeze dried in trehalose, inulin and skim milk maintained the highest acidi~catiort 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. lactic BDBB2 when cultured in SPY 2 (Figure 6). This observation remains unexplained, as cells would not .. -26-have 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.
Iflrobiotic inhibition of pathogens The pathogen inhibition trials area 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 el~eets of -1o 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 nat shown} on the buffered media described by Chateau et al. (1993}.
~. coli and ~.. monocytogenes rxrere 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.
:~isteria monacytogenes, has been examined for inhibition by probiotic organisms, along 2o with the observation from this work that Lisreria 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 of E. cola and L. manoeytogenes.
B. lactis HDBB2 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 BABB2 was tested on RC.A., containing only a quarter o~the amount of glucose of MRS. L. acidophilus M,1L,A.1 and L.
rhamnosus i..CST~l are able to ferment trehalose, whereas B, lactis BDB$2 can not, so the trehalose 3o cryoproteetant can serve as an extra carbohydrate source for those strains-None ofthe strains are able to utilise inuliri as a carbohydrate source, despite inulin being described as a 'bifidogenic' substance (Roberfroid, 1993), Alternatively, pathogen inhibition may not be related to acid production.
30uD'llD2,uc122l5spea426 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 ,t~'. colt by either l Omm or 14 mm is questionable-Optimisation of cryoprotectant There were differences in the ability of each cryoprotectant to prevent cell 1o deactivation and freeze injury duriztg 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 fxeeze drying stage.
is 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 MJL.A,>l 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 2o 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 (15%), trehalose (15%), inulin (7.5%)/trehalose (7.5%) or trehalose (15%)/ascorbic acid {4 mglL). Ascorbic acid has been shown to 25 improve survival o~L. 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 tlae storage experiment.
The use of either trehalose or-inulin as cryoprotectant for probiotic organisms has proven to be better than the reference cryoprotectam skim milk (10°/a), at preventing initial 3o deactivation and sub lethal injury, and nnaintaining cell viability during storage. Increasing the cryoprotectant concentration to 15% may further improve these results, by increasing the survival of the freeze drying process.

' _ .
Trehalose (a-D-glucopyranosyl-D-glucopyranose) has been reported to be an effective cryoprotectant for yeasts and bacteria. Trehalose litre other disaccharides, is thought to be an effrective 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 l, 3300 Tienen, Belgium), make inulin an excellent replacement far skim milk.
to 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 nnieroorga,nisms as set forth in Example 1.
1~'roaen soy desserd All product ingredients (soy beverage, sugar, oil, stabiliser and salt) are combined and heated to 50°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 Il Gelataio 1 b00). The frozen soy dessert can then be packed z5 into containers, sealed and hardened to -20°C: The samples axe to be stored at -20°C.
The product pH is 7.0 +I- 0.2.
The cryoprotection ability of inulin in the product can also be increased, by addition of extra inulir~ 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.

Yoghurt The base ingredients of milk and mills powder (S%) are combined and heated to 85°C for 30min and tempered to 43°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 cfulmL. The yoghurt base is then incubated at 43°C until a ply of4.5 has been achieved. Freeze dried probiotic microorganisms, such as L, acidophilus M1LA1 freeze dried in inulin, are added to the mix at a concentration of 106 cfulrnL and evenly dispersed. The base is then dispensed into plastic tubs, sealed and stored at 4°C.
1~ Bxtxa inulin (up to 2%) can be included in the yoghurt base to aid cryoprotection of probiodc 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.

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Claims (44)

The claims defining the invention are as follows:-

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.

5. 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.

10. 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.

11. A method for entrancing 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 of topinambour, chicory, onion, asparagus and artichoke.

15. 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.

20. 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.

25. 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.

30. 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.

40. Use of a microorganism according to any one of claims 35 to 38 for incorporation into a food.

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 wore 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.