ABALONE

BACKGROUND OF THE INVENTION

This invention relates to culture media and processes for producing probiotic microorganisms for use in the aquaculture of abalone using a novel bioprocess technology.

A bioprocess for the production of abalone probiotics does not currently exist. This invention facilitates the bioproduction of abalone probiotics from a small volume cryo-culture of Vibrio midae a bacterial probiotic, or Debaryomyces hansenii a yeast probiotic or a combination thereof into probiotic products. The invention describes the culture media, inoculum procedures, the fermentation processes, harvesting of the biomass and formulation of the biomass into liquid and/or dry products which can be incorporated into abalone feed.

Abalone (family Haliotidae); are marine vestigastropod mollusks with a worldwide distribution in coastal temperate and tropical waters (Degnan et a/., 2006). The abalone family consists of about 56 species all belonging to the genus Haliotis. Abalone are one of the most valuable seafood species in the world (Reddy-Lopata et al., 2006) and are in considerable demand in the Far East where they have been exploited for many years (Francis et al., 2007). More often than not demand for abalone far exceeds supply and natural abalone stocks are under severe pressure due to illegal harvesting and poaching. The flesh of certain large abalones is considered by many to be a desirable food and is considered one of the most expensive protein products in the world. The high value has promoted the development and optimization of intensive abalone aquaculture (Park et al., 2007). However, abalone aquaculture is challenged by factors, such as poor water quality, disease and the intrinsic slow growth rate of this marine mollusc.

The successful aquaculture of abalone relies upon proper nutrition and elimination of diseases (Naidoo et al., 2006). The conventional solution to this problem has been the use of antimicrobial drugs in the aquaculture of abalone, this approach has however led to the development of antibiotic resistant bacteria (Schwarz et al., 2001), causing a negative impacts on the environment and the cultivation of abalone, as well as consumer resistance. Macey and Coyne (2006) suggested that a probiotic-supplemented diet improves growth and the immune system of abalone.

Abalone, has an extremely slow growth rate, and takes approximately four years to reach a size that is market acceptable. It was therefore imperative to investigate methods to enhance the growth rate of abalone, as well as to increase its tolerance to disease

An alternative approach to the challenges facing abalone aquaculture is the use of probiotic microorganisms. This technology is gaining popularity due to an increasing demand for environmentally friendly aquaculture. Probiotics are live microorganisms which are administered in adequate amounts to confer a health effect on the host. They have been shown to control diseases, improve digestion and boost overall growth and immunity of the cultured species.

Debaryomyces hansenii and Vibrio midae were isolated from the gastrointestinal tract of the abalone, Haliotis midae, by Macey and Coyne (2006) and evaluated for probiotic activity, specifically the ability to increase the growth rate of abalone. The studies conducted by Macey and Coyne (2005) showed that supplementation of abalone diet with these probiotic microorganisms led to an improved growth rate. The probiotic microorganisms were able to break down complex polysaccharides and proteins present in the natural seaweed diets of the abalone and were capable of enhancing digestive efficiency, thereby improving the growth rate of the abalone (Coyne et al., 2004). These microorganisms increase certain activities within the gut of abalone, like phagocytic activity, protease activity, and amylase activity.

The state of the art describes the use of naturally occurring bacteria and yeast as probiotics in aquaculture. In this regard, the prior art discloses the use of bacteria from the family Vibrionaceae as probiotics for use in the aquaculture of prawns (Ruangpan et al., 1998); and shrimp (Austin et al., 1995). Other Vibrio species isolated from marine environments have also displayed probiotic effects and have been reported by Nair et al., 1985; Carraturo ef al., 2006 and Castro et ^' al., 2006.

South African patent 2004/06777 and the work performed by Macey and Coyne (2005) and (2006), describe the use Vibrio midae and Debaryomyces hansenii as probiotics for use in abalone aquaculture. In this body of work Vibrio midae was cultured in marine broth (MB) at 22°C and Debaryomyces hansenii was cultured in yeast extract-peptone-glucose broth (YPD) medium at 22°C. The work performed by Macey & Coyne did not investigate optimal culture conditions, cell culture concentrations, measure optical density of the cultures and/or measure the production of biomass. A comparative assessment of the processes and production media utilised by Macey and Coyne and processes and production media of the present invention reveal that the inventors have been able to significantly increase cell productivity to several orders of magnitude greater than that described in the prior art for both Vibrio midae and Debaryomyces hansenii (see Example 3).

Debaryomyces hansenii is commonly used as an industrial yeast. Previous studies in respect of this yeast have generally not focussed on its use as a whole cell biocatalyst, but rather on its use for the production of metabolites, such as glycerol and xylose (Adler and Gustafsson, 1980 and Parajo et al., 1997). The prior art does, however describe Debaryomyces hansenii as a member of the microbiota of aquatic species, such as seabass (Tovar et al., 2002) and rainbow trout (Andlid et al., 1995). In these studies Debaryomyces hansenii was also cultivated in a yeast extract-peptone-glucose broth (YPD) and neither of these studies investigated the optimal culture conditions, cell culture concentration, optical density or biomass production.

The present specification describes a novel high productivity bioprocesses for the cultivation of Vibrio midae and Debaryomyces hansenii and their use in aquaculture. No previous work in this field provides a process for large scale production of abalone probiotics.

SUMMARY OF THE INVENTION

The present invention provides for a novel process for producing a probiotic feed composition, wherein the probiotic feed composition comprises live cultures of Vibrio midae and/or Debaryomyces hansenii cells and/or combinations of the live cultures.

According to a first aspect of the invention there is provided for a growth medium for culturing probiotics for use in the production of probiotic compositions and probiotic feeds for abalone.

In one embodiment of the invention the growth medium is a bacterial growth medium comprising citric acid, H ₃ P0 ₄ , from about 0 g/l to about 4 g/l (NH ₄ ) ₂ S0 ₄ , Ca(N0 ₃ ) _2, MnS0 ₄ .7H ₂ 0, FeS0 ₄ .7H ₂ 0, KCI, from about 20 g/l to about 40 g/l NaCI, MgCI ₂ .6H ₂ 0, from about 42 g/l to about 255g/l corn steep liquor, from about 1 g/l to about 40 g/l inverted high test molasses and from about 0.5 ml/I to about 5 ml/I antifoaming agent; and the pH of the growth medium is from about 5 to about 8. Preferably, the bacterial growth medium comprises about 1 g/l citric acid, about 2.5 ml/l H ₃ PO4, about 0.4 g/l Ca(N0 ₃ ) ₂ , about 0.040 g/l MnS0 ₄ .7H ₂ 0, about 0.032 g/l FeS0 ₄ .7H ₂ 0, about 1 g/l KCI, about 30 g/l NaCI, about 2.3 g/l MgCI ₂ .6H ₂ 0, about 54.4 g/l corn steep liquor, about 24 g/l inverted high test molasses; and about 1 ml/l antifoaming agent; and wherein the pH of the growth medium is maintained at about 6.5. The bacterial growth medium supports significantly higher cell productivity (g/l/h) of Vibrio midae than Marine broth (MB) medium. In another embodiment of the invention the growth medium is a yeast growth medium comprising citric acid, H ₃ P0 ₄ , from about 18g/l to about 30g/l (NH ₄ ) ₂ S0 ₄ , MgS0 ₄ .7H ₂ S0 ₄ , CaCI ₂ .2H ₂ 0, from about 0 g/l to about 40 g/l NaCI, KH ₂ P0 ₄ , from about 8 g/l to about 212 g/l corn steep liquor, from about 1 g/l to about 40 g/l inverted high test molasses; and from about 0.5 ml/l to about 5 ml/l antifoaming agent; and the pH of the growth medium is from about 4 to about 7. Preferably the yeast growth medium comprises about 2.5 g/l citric acid, about 16.3 ml/l H ₃ P0 ₄ , about 30 g/l (NH ₄ ) ₂ S0 ₄ , about 8.2 g/l gS0 ₄ .7H ₂ S0 ₄ , about 0.8 g/l CaCI ₂ .2H ₂ 0, about 0.1 g/l NaCI, about 1 1.3 g/l KH ₂ P0 , about 204 g/l corn steep liquor, about 10 g/l glucose; and about 1 ml/l antifoaming agent; and wherein the pH of the growth medium is maintained at about 5.6. The yeast growth medium supports significantly higher cell productivity (g/l/h) of Debaryomyces hansenii than the yeast extract-peptone-glucose (YPD) medium.

According to a second aspect of the invention there is provided for a process for producing a probiotic composition, wherein the process comprises the steps of (i) preparing a pre-fermentation culture by inoculating an inoculum medium with a culture of live Vibrio midae cells and/or live Debaryomyces hansenii cells, (ii) inoculating a growth medium contained in a reactor with the pre-fermentation culture, (iii) controlling airflow, oxygen saturation, back pressure, temperature and pH of the growth medium during the growth phase of the Vibrio midae cells and/or Debaryomyces hansenii cells, (iv) harvesting the Vibrio midae cells between the mid- log and stationary phase of growth, when the cells are in a stable form and when the live cell concentration is in the range of 2x10 ⁸ cells/ml to 1 x10 ¹¹ cells/ml . Alternatively, harvesting the Debaryomyces hansenii cells between the mid-log and late-log phase of growth when the live cell concentration is in the range of 4x10 ⁷ cells/ml to 2x10 ¹⁰ cells/ml; and (v) producing a liquid product by resuspending the harvested Vibrio midae cells and/or Debaryomyces hansenii cells in a buffer.

The isolated Vibrio midae described in this specification was deposited with The Belgian Coordinated Collections of Micro-organisms (BCCM) on 1 July 2013 under Accession No. LMG P-27727. Similarly, cultures of the isolated Debaryomyces hansenii described in this specification were deposited on 1 July 2013 under Accession No. MUCL 54982. The process may further comprise the steps of (vi) blending the liquid product with a carrier, stabiliser or filler to form a biomass paste, and (vii) drying the biomass paste to produce a dry product.

The process of the invention may be a batch process or a fed-batch process. Preferably the process is a batch process for Vibrio midae and a fed-batch process for Debaryomyces hansenii.

In a preferred embodiment of the invention the airflow into the reactor for the fed batch or batch process may be in the range of 0.5 to 2.0 v/v/m. Preferably, the airflow into the reactor is 1 v/v/m.

In a further preferred embodiment of the invention the dissolved oxygen in the fermentation medium is preferably in the range of 20 to 100% saturation. More preferably the dissolved oxygen is maintained at 30% saturation.

In yet a further preferred embodiment of the invention the backpressure in the reactor is preferably in the range of 0 to 250 kPa. Most preferably the backpressure in the reactor is 50 kPa.

The Vibrio midae cells are preferably cultured in the culture medium at a temperature in the range of 20 to 34°C. Most preferably the Vibrio midae cells are cultured at a temperature of 30°C. On the other hand, the Debaryomyces hansenii cells are preferably cultured in the culture medium at a temperature in the range of 16 to 34°C. Most preferably the Debaryomyces hansenii cells are cultured at a temperature of 24°C.

The pH of the culture medium for Vibrio midae is preferably in the range of 5.0 to 8.0, and most preferably the pH is 6.5. The pH of the culture medium for Debaryomyces hansenii is preferably in the range of 4.0 to 7.0, and most preferably the pH is 5.6.

It will be appreciated that the growth medium used in the process of the invention comprises nutrients for the growth of the Vibrio midae and/or Debaryomyces hansenii cells. The nutrients are selected from the group consisting of inorganic salts, sources of carbon, nitrogen, vitamins, trace elements, antifoam agents and mixtures thereof. Further, it will be appreciated that the inoculum medium comprises nutrients for the growth of the probiotics used in the process of the invention selected from the group consisting of inorganic salts, sources of carbon, nitrogen, vitamins, trace elements and mixtures thereof.

In one embodiment of the invention the probiotic microorganisms are harvested from the growth medium by centrifugation or filtration. In a preferable embodiment the probiotic microorganisms are harvested by vertical tube centrifugation.

In a preferred embodiment of the invention the Vibrio midae probiotic microorganisms are re-suspended in a saline phosphate buffer to produce a liquid product. Similarly, the Debaryomyces hansenii probiotic microorganisms are re- suspended in a phosphate buffer to produce the liquid product, preferably the phosphate buffer will include from about 4-20% m/m trehalose, preferably 12.5% m/m trehalose; and the pH of the buffer will be in the range of about 4 to 6, preferably the pH is 4.5.

In a further preferred embodiment of the invention the liquid product is combined with a carrier selected from the group consisting of micro-crystalline cellulose, trehalose, sucrose and skimmed milk or combinations thereof to form a blended product. In yet another preferred embodiment the biomass paste is extruded and dried to form a dry product.

Extrusion of the Vibrio midae liquid product in combination with the carrier will typically be performed at a temperature in the range of 30 to 70°C, however preferably extrusion will take place at a temperature of 45°C. Extrusion of the Debaryomyces hansenii liquid product in combination with the carrier will typically be performed at a temperature in the range of 30 to 50°C, however preferably extrusion will take place at a temperature of 45°C. According to a third aspect of the invention there is provided for a probiotic composition comprising the liquid product or dry product manufactured according to the process of the invention and containing Vibrio midae cells and/or Debaryomyces hansenii cells and/or combinations of the two for use in the aquaculture of abalone.

In a preferred embodiment of the invention the liquid product or dry product made according to the process of the invention is formulated into abalone feed. Further, the liquid product or dry product may be combined with the abalone feed by pre-blending prior to extrusion of the feed, vacuum infusion into pre-prepared abalone feed or by top coating existing feed.

Extrusion of the Vibrio midae liquid or dry product in combination with abalone feed will typically be at a temperature in the range of 30 to 70°C, however preferably extrusion will take place at a temperature of 45°C. Extrusion of the Debaryomyces midae liquid or dry product in combination with abalone feed will typically be at a temperature in the range of 30 to 70°C, however preferably extrusion will take place at a temperature of 45°C.

Yet another aspect of the invention provides for a probiotic composition for abalone which comprises a combination of abalone feed with the Vibrio midae probiotic composition and the Debaryomyces hansenii probiotic composition of the invention.

BRIEF DESCRIPTION OF THE FIGURES

Non-limiting embodiments of the invention will now be described by way of example only and with reference to the following figures:

Figure 1a: Cell productivity (CFU/ml/h) data obtained during the cultivation of

Vibrio midae in a 2L Braun B bioreactor over a range of temperature conditions (10°C to 40°C).

Figure 1 b: Cell productivity (CFU/ml/h) data obtained during the cultivation of

Debaryomyces hansenii in a 2L Braun B bioreactor over a range of temperature conditions (10°C to 40°C). Figure 2a: Cell productivity (CFU/ml/h) data obtained during the cultivation of Vibrio midae in a 2L Braun B bioreactor over a range of pH conditions (5.0 to 8.0).

Figure 2b: Cell productivity (CFU/ml/h) data obtained during the cultivation of

Debaryomyces hansenii in a 2L Braun B bioreactor over a range of pH conditions (4.0 to 7.0).

Figure 3a: Cell productivity of Vibrio midae, measured during the cultivation of the organism in fermentation medium containing various concentrations of CSL in 15L Braun C bioreactors.

Figure 3b: Cell productivity of Debaryomyces hansenii, measured during the cultivation of the organism in fermentation medium containing various concentrations of CSL in 15L Braun C bioreactors.

Figure 4a: Cell productivity of Vibrio midae, measured during the cultivation of the organism in fermentation medium containing various concentrations of TSAI in 15L Braun C bioreactors.

Figure 4b: Cell productivity of Debaryomyces hansenii, measured during the cultivation of the organism in fermentation medium containing various concentrations of TSAI in 15L Braun C bioreactors.

Figure 5: Growth of Vibrio midae in an in-house developed growth medium to achieve a high cell density production of the organism

Figure 6: Fermentation profiles depicting operating parameters for growth of

Vibrio midae in optimum fermentation medium in a 15L Braun C bioreactor.

Figure 7: Key responses obtained and evaluated during the cultivation of Vibrio midae in media supplemented with 54.4 g/l CSL and 25 g/l HTM. Figure 8: Growth of Debaryomyces hansenii in an in-house developed growth medium to achieve a high cell density production of the organism.

Figure 9: Fermentation profiles depicting operating parameters for growth of

Debaryomyces hansenii in optimum fermentation medium in a 15L Braun C bioreactor.

Figure 10: Key responses obtained during the cultivation of Debaryomyces hansenii strain in media supplemented with 24 g/l CSL and 10 g/l HTM.

DETAILED DESCRIPTION OF THE INVENTION

The present invention will now be described more fully hereinafter with reference to the accompanying drawings, in which some, but not all embodiments of the invention are shown.

The invention as described should not to be limited to the specific embodiments disclosed and modifications and other embodiments are intended to be included within the scope of the invention. Although specific terms are employed herein, they are used in a generic and descriptive sense only and not for purposes of limitation.

The use of the word "comprise" and variations of the word, such as "comprising" and "comprises," means "including but not limited to," and is not intended to exclude, for example, other additives, components, integers, or steps.

As used herein, the term "live bacterial cell" or "live yeast cell" refers to a microorganism that is alive and capable of multiplication and/or propagation or to a microorganism which is viable but not culturable, while in a vegetative, frozen, preserved, or reconstituted state.

As used herein, the term "viable cell yield" or "viable cell concentration" refers to the number of viable cells in a liquid culture, concentrated, or preserved state, and may include live bacterial and/or yeast cells which are viable but not culturable, per unit of measure, such as liter, milliliter, kilogram, gram or milligram. As used herein, the term "cell preservation" refers to a process that takes a vegetative cell and preserves it in a metabolically inert state that retains viability over time. As used herein, the term "product" refers to a microbial composition that can be blended with other components and contains specified concentration of viable cells that can be sold and used.

As used herein, the term "probiotic" refers to one or more live microorganisms that confer beneficial effects on a host organism. Benefits derived from the establishment of probiotic microorganisms within the digestive tract include reduction of pathogen load, improved microbial fermentation patterns, improved nutrient absorption, improved immune function, and aided digestion.

As used herein, the term "fermentation" refers to a metabolic process performed by an organism that converts one substrate to another in which the cell is able to obtain cellular energy, such as when an organism utilizes glucose and converts it to lactic acid or propionic acid. In the present application the fermentation process is used to produce cellular energy in order to facilitate reproduction of the bacterial and/or yeast strains and thus produces more of these microorganisms.

The process of the invention provides for the production of an abalone feed comprising the bacterium Vibrio midae or the yeast Debaryomyces hansenii or combinations of the two.

The probiotic is produced by replication of these microorganisms in a high productivity fermentation process.

The fermentation process of the invention may be a batch process or fed- batch process. However, the process is preferably a batch process for Vibrio midae and a fed-batch process for Debaryomyces hansenii.

The fermentation airflow is in the range 0.5 to 2.0 v/v/m and preferably 1 v/v/m for either Vibrio midae or Debaryomyces hansenii. The fermentation agitation speed is controlled to maintain the dissolved oxygen in the range 20 to 100% saturation, preferably at 30% saturation for either Vibrio midae or Debaryomyces hansenii. The fermentation oxygen utilization rate is in the range 10 to 210 mMol/l/h for either Vibrio midae or Debaryomyces hansenii and nominally 160 mMol/l/h for Vibrio midae and 200 mMol/l/h for Debaryomyces hansenii.

The fermentation backpressure is in the range 0 to 250kPa and optimally the fermentation backpressure is 50kPa.

The fermentation temperature is in the range 20 to 34°C with an optimum of 30°C for Vibrio midae (Figure 1 a) and in the range 16 to 34°C with an optimum of 24°C for Debaryomyces hansenii (Figure 1 b).

The fermentation pH is in the range 5.0 to 8.0 with an optimum of 6.5 for Vibrio midae (Figure 2a) and in the range .4.0 to 7.0 with an optimum of 5.6 for Debaryomyces hansenii (Figure 2b). The fermentation pH is controlled using an acid or base. The acid is preferably sulphuric acid and the base is preferably ammonium hydroxide.

The growth medium comprises salts, preferably the following for Vibrio midae (magnesium chloride hexahydrate, potassium chloride, citric acid, calcium nitrate, manganous sulphate tetrahydrate, iron sulphate heptahydrate and phosphoric acid) and (citric acid, magnesium sulphate heptahydrate, calcium chloride dehydrate, potassium dihydrogen prthophosphate and phosphoric acid) for Debaryomyces hansenii.

The growth medium comprises a source of inorganic nitrogen, NaCI, antifoam agent, carbohydrates and an organic nutrient source. The inorganic nitrogen source may be ammonium sulphate in the range 0 to 15 g/l, preferably 0 to 4 g/l, most preferably 0 g/l for Vibrio midae and in the range 15 to 50 g/l, preferably 18 to 30, and most preferably 18.5 g/l for Debaryomyces hansenii. The source of NaCI is in the range 10 to 50 g/l, preferably 20 to 40 g/l, most preferably 30 g/l for Vibrio midae and in the range 0 to 10 g/l, preferably 0.1 g/l for Debaryomyces hansenii. The growth medium comprises of an antifoam agent in the range 0.5 to 10 ml/I, preferably 1 to 5 ml/l, most preferably 1 ml/I. The organic nutrient source may be Corn Steep Liquor (hereinafter referred to as "CSL"), peptone, yeast extract and or casamino acids, preferably liquid phytase treated and ultra-filtered CSL in the range 42 to 255 g/l with an optimum concentration of 54.4 g/l for Vibrio midae (Figure 3a) and in the range of 8 to 212 g/l with an optimum concentration of 204 g/l for Debaryomyces hansenii (Figure 3b). The carbohydrate source may be glucose, fructose, sucrose or molasses, but is preferably inverted high test molasses (hereinafter referred to as "HTM") in the range 1 to 40 g/l total sugar as invert (herein after referred to as "TSAI") with an optimum of 24 g/l for Vibrio midae (Figure 4a) and 1 to 40 g/l TSAI (glucose or HTM) with and optimum of 10 g/l for Debaryomyces hansenii (Figure 4b).

The micro-organisms are grown in an inoculum stage to improve productivity and robustness in an inoculum medium similar to the growth medium for Vibrio midae (magnesium chloride hexahydrate, potassium chloride, citric acid, calcium nitrate, manganous sulphate tetrahydrate, iron sulphate heptahydrate, phosphoric acid, yeast extract, casamino acids and peptone) or (citric acid, magnesium sulphate heptahydrate, calcium chloride dehydrate, potassium dihydrogen orthophosphate, phosphoric acid and yeast extract) for Debaryomyces hansenii.

The fermentation productivity is in the range 1.0x10 ⁸ to 1.6x10 ¹³ cells/l/h with an optimum of 1.5x10 ¹³ cells/l/h for Vibrio midae and 3.0x10 ⁰⁴ to 7.0x10 ¹¹ cells/l/h with an optimum of 6.4x10 ¹ cells/l/h for Debaryomyces hansenii.

The fermentation microorganism cell concentration is in the range 2x10 ⁸ to 1x10 ¹¹ cells/ml with an optimum of 9.2x10 ¹⁰ cells/ml for Vibrio midae and 4.0x10 ⁷ to 2.0x10 ¹⁰ cells/ml with an optimum of 1.6x10 ¹⁰ cells/ml for Debaryomyces hansenii.

The cultured Vibrio midae or Debaryomyces hansenii are harvested from the fermentation broth by centrifugation or filtration, preferably vertical tube centrifugation and/or continuous centrifugation.

The biomass may be formulated into a suitable buffer, preferably phosphate buffer containing NaCI to yield a probiotic liquid product. Alternatively, for Debaryomyces hansenii the phosphate buffer includes from about 4-20% m/m trehalose, preferably 12.5% m/m trehalose; and the pH of the buffer is in the range of about 4 to 6, preferably the pH is 4.5. The liquid product form may be formulated with a dry carrier, stabilizer or filler such as micro-crystalline cellulose (herein after referred to as "MCC"), trehalose, sucrose or skimmed milk, preferably a combination of trehalose and MCC into a biomass paste.

The biomass paste may be extruded and dried to yield a probiotic dry product. The biomass paste may be extruded at a temperature in the range of 30 to 50°C, but preferably the biomass paste is extruded at a temperature of 45 °C.

The probiotic liquid product or probiotic dry product may be formulated into abalone feed, by pre-blending prior to extrusion of the feed, or by vacuum infusion into existing feed or by top-coating of existing feed.

No evidence has been found to suggest that the organisms described herein have been produced in high cell density cultures or at a significantly higher cell productivity (g/h/l) than described in the prior art. Further, standard processes typically yield cell titres of 1x10 ⁹ cells/ml, surprisingly using the processes described in this specification produce significantly higher cell titres (1.57x10 ¹⁰ cells/ml of Debaryomyces hansenii and 9.72x10 ¹⁰ cells/ml of Vibrio midae).

The following examples are offered by way of illustration and not by way of limitation.

EXAMPLE 1

Production of Vibrio midae abalone probiotic

The Vibrio midae described herein is known to enter a viable but not culturable (VNBC) state. The processes and media described herein however allow V. midae to enter into a culturable state for the purposes of large scale production of these probiotics.

Vibrio midae inoculum

A single cryovial of Vibrio midae was used to inoculate each inoculum flask (1.81 Fernbach). The growth media in each flask consisted of: 1 g/l citric acid, 2.5 ml/l H3PO4, 3 g/l (NH ₄ ) ₂ S0 ₄ , 0.4 g/l Ca(N0 ₃ ) ₂ , 0.04 g/l MnS0 ₄ .7H ₂ 0, 0.032 g/l FeS0 ₄ .7H _z O, 1 g/l KCI, 30 g/l NaCI, 2.3 g/l MgCl _z .6H ₂ 0, 5 g/l casamino acids, 5 g/l yeast extract, 10 g/l peptone (Biolab) and 10 g/l glucose. The pH of the growth media was adjusted to 6.5, using 10% v/v H ₂ S0 ₄ or 25% v/v NH ₄ OH, prior to sterilization at 121 °C for 15 min. Following inoculation, the flasks were incubated at 30°C at a speed of 180 rpm in an orbital shaker (Innova 2300, New Brunswick Scientific, Edison, NJ, USA) for 4.5 h (OD _660nm ~ 2.0).

Vibrio midae fermentation process

A single flask of inoculum, was used to inoculate each fermenter. The fermentation process was conducted in 15 I Biostat C™ fermenters (Sartorius BBI systems, Melsungen, Germany) at a working volume of 10 I.

The growth media used in the fermenters contained the following medium components: 1 g/l citric acid, 2.5 ml/l H ₃ P0 ₄ , 0.4 g/l Ca(N0 ₃ ) ₂ , 0.040 g/l MnS0 ₄ .7H ₂ 0, 0.032 g/l FeS0 ₄ .7H ₂ 0, 1 g/l KCI, 30 g/l NaCI, 2.3 g/l MgCI ₂ .6H ₂ 0, 54.4 g/l CSL and 24 g/l HTM. The growth media salts, antifoam (1 ml/l Pluriol™ P2000, BASF, Ludwigshafen, Germany) and CSL were added to the initial charge and made up to 9.3 I. Following the sterilization of the initial charge, a separately sterilized HTM solution was added. The fermentation temperature was maintained at 30°C. The stirrer speed was set at 500 rpm and ramped to 1000 rpm, to maintain the dissolved oxygen above 30% saturation. The pH was maintained at 6.5 using 10% v/v H ₂ S0 ₄ or 25% v/v NH ₄ OH. Aeration was set at 1 v/v/m.

Down-stream processing of Vibrio midae strains

The fermentation broth was centrifuged using a Sharpies-Stokes centrifuge (Pennwalt, France). The centrifuge tube speed was 15700 g with a fermentation broth feed rate of 30 l/h. The supernatant was discarded and the biomass was formulated with a phosphate buffer (KH ₂ P0 ₄ 0.11 , K ₂ HP0 ₄ 0.71 , NaCI 2.91 , H ₂ 0 96.27% m/m). An overhead stirrer (paddle agitator) was used to homogenize the liquid product (1000 rpm).

For the extruded dry product form, the bacterium (formulated in saline phosphate buffer), was blended in a food mixer, KMX50 (Kenwood, London, UK), with MCC (1 :1 liquid product to MCC by mass) and trehalose (5% m/m). The blend was cold extruded through a sieve-type extruder, Nica E-140 extruder (Niro- Aeromatic, Southampton, UK) and then convection dried to ~10% m/m using a bench scale Aeromatic Fluidised Bed Dryer (Aeromatic AG, Germany). The inlet airflow was set at 50 m /h and at ambient temperature.

For the abalone feed, the centrifuged biomass was re-suspended in an artificial salt water (ASW) buffer. The re-suspended buffer was added to abalone feed and cold extruded using a screw extruder and dried in a convection oven (at 30°C) to a final moisture content of -10% m/m. The re-suspended biomass was extruded at a temperature in the range of 30 to 70°C. However, preferably the biomass paste is extruded at a temperature of 45°C.

Results

A maximum cell concentration of 9.7x10 ¹⁰ cells/ml and optical density of 11.1 was obtained when Vibrio midae were cultivated in medium containing 24 g/l HTM and 54.4 g/l CSL after 6 hours of cultivation (Figure 5).

Airflow and temperature was maintained at 1 v/v/m and 30°C throughout the fermentation. pH was controlled automatically at 6.5 using 10% v/v H ₂ S0 ₄ or 25% v/v NH ₄ OH. P0 ₂ was maintained above 30% by ramping up agitation speed when necessary. A maximum oxygen utilization rate (OUR) of 160 mMol/l/h was achieved in this fermentation (Figure 6).

The key responses measured were growth rate, cell concentration, cell productivity and cell yields on protein (YPP), sugar (YPS) and oxygen (YPO) (Figure 7).

A growth rate of 1.0/h was observed. A maximum cell concentration of 9.7x10 ¹⁰ cells/ml and cell productivity of 1.6x10 ¹³ cells/ml/h was obtained by cultivating Vibrio midae in the fermentation medium. Vibrio midae cell yields on protein, sugar and oxygen were 1.8x10 ¹³ cells/g, 4.1x10 ¹² cells/g and 2.0x10 ¹⁰ cells/g respectively (Figure 7).

The liquid product has a half-life of 100 days (storage at 4°C). Formulated to a final product initial count of 2x10 ¹⁰ CFU/ml, the shelf-life is 669 days (to a final product count of 1x10 ⁸ CFU/ml). The extruded product has a half-life of 4.86 days (storage at 4°C). Formulated to a final product initial count of 2x10 ¹⁰ CFU/ml, the shelf-life is 31 .5 days (to a final product count of 1x10 ⁸ CFU/ml).

The abalone feed has a half-life of 22.4 days (storage at 4°C). Formulated to a final product initial count of 2x10 ¹⁰ CFU/ml, the shelf-life is 156.8 days (to a final product count of 1x10 ⁸ CFU/ml).

Limited information was previously available relating to the growth, cultivation and maintenance of Vibrio midae. There is only one piece of literature that confers information on the growth of this organism, and that is provided by the individuals who isolated the organism (Macey and Coyne, 2006). In their publication they teach the cultivation of Vibrio midae in a standard Marine broth-MB at a temperature of 22°C and pH of -7.0.

On the other hand the present invention provides for a bacterial growth medium, which led to a 27% increase in the productivity of Vibrio midae and a substantial reduction in the cost of production (109108%).

Further, the teachings of Macey and Coyne, 2006 suggest an optimal growth temperature of 22°C, however it was found that increasing the temperature to 30°C led to a 1077-fold improvement in cell productivity. Furthermore, by adjusting the pH to, a slightly more acidic, 6.5 resulted in a further improvement of 333% in cell productivity when used together with the bacterial growth media described.

By using CSL as a nutrient source, at optimum pH and temperature, the performance of the process was significantly improved and resulted in a surprising 24% increase in cell concentration, a 2750-fold increase in cell productivity, and a 1.82x10 ⁶ % decrease in the cost of production.

Further, by including HTM as a carbon source the process yielded an increase in cell concentration of 181 %, a 5705-fold increase in cell productivity and a 3.74x10 ⁶ % decrease in cost of production. The combination of the abovementioned factors has led to the development of a novel process for the production of Vibrio midae as a probiotic with significant increases in productivity, concentration and yield, coupled with a substantial reduction in the cost of production of this organism.

The cumulative effect of culturing Vibrio midae in the culture medium described herein and using the process of this example has resulted in a 31 17-fold increase in productivity over the previous production process. This increased output may be attributed to the new growth medium, together with the use of an optimal temperature and pH. The productivity of the process was further improved by a further 83%, by substituting commonly used nitrogen and carbohydrate sources with CSL and HTM. A 99% decrease in cost was observed at the final process optimization step. This study provides a vast amount of new knowledge regarding the cultivation of Vibrio midae.

EXAMPLE 2

Production of Debaryomvces hansenii abalone probiotic Debaryomyces hansenii inoculum

A single cryovial of Debaryomyces hansenii (5.9x10 ⁸ CFU/ml) was used to inoculate each inoculum flask (1.81 Fernbach). The growth media in each flask consisted of: 2.5 g/l citric acid, 16.3 ml/I H ₃ P0 ₄ , 30 g/l (NH ₄ ) ₂ S0 ₄ , 8.2 g/l MgS0 ₄ .7H ₂ S0 ₄ , 0.8 g/l CaCI ₂ .2H ₂ 0, 0.1 g/l NaCI, 11.3 g/l KH ₂ P0 ₄ , 10 g/l yeast extract and 10 g/l glucose. The pH of the growth media was adjusted to 5.6, using 10% v/v H ₂ S0 ₄ or 25% v/v NH ₄ OH, prior to sterilization at 121 °C for 15 min. Following inoculation, the flasks were incubated at 24°C at a speed of 180 rpm in an orbital shaker (Innova 2300, New Brunswick Scientific, Edison, NJ, USA) for 22 h (ODeeonm ~ 4.0).

Debaryomyces hansenii fermentation process

A single flask, of inoculum, was used to inoculate each fermenter. The fermentation process was conducted in 15 I Biostat C™ fermenters (Sartorius BBI systems, Melsungen, Germany) at a working volume of 10 I. The growth media used in the fermenters contained the following medium components (g/l); 2.5 g/l citric acid, 16.3 ml/l H ₃ P0 ₄ , 30 g/l (NH ₄ ) ₂ S0 ₄ , 8.2 g/l MgS0 ₄ .7H ₂ S0 ₄ , 0.8 g/l CaCI ₂ .2H ₂ 0, 0.1 g/l NaCI, 11.3 g/l KH ₂ P0 ₄ , 204 g/l CSL and 10 g/l glucose or HTM. The growth media salts, antifoam (1 ml/l, Pluriol™ P2000, BASF, Ludwigshafen, Germany) and CSL were added to the initial charge and made up to 9.3 I. Following the sterilization of the initial charge, a separately sterilized HTM solution was added. The fermentation temperature was maintained at 24°C. The stirrer speed was set at 500 rpm and ramped to 1000 rpm, to maintain the dissolved oxygen above 30% saturation. The pH was maintained at 5.6 using 10% v/v H ₂ S0 or 25% v/v NH ₄ OH. Aeration was set at 1 v/v/m.

Down-stream processing of Debaryomyces hansenii strains

The fermentation broth was centrifuged using a Sharpies-Stokes centrifuge (Pennwalt, France). The centrifuge tube speed was 15700 g with a fermentation broth feed rate of 30 l/h. The supernatant was discarded and the biomass was formulated with a phosphate buffer (KH ₂ P0 ₄ 0.11 , K ₂ HP0 ₄ 0.71 , NaCI 2.91 , H ₂ 0 96.27 %m/m). An overhead stirrer (paddle agitator) was used to homogenize the liquid product (1000 rpm).

For the extruded dry product form, the Debaryomyces hansenii (formulated in liquid phosphate buffer), was blended in a food mixer, KMX50 (Kenwood, London, UK), with MCC (1 :1 liquid product to MCC by mass) and trehalose (5% m/m). The blend was cold extruded through a sieve-type extruder, Nica E-140 extruder (Niro- Aeromatic, Southampton, UK) and then convection dried to ~10% m/m using a bench scale Aeromatic Fluidised Bed Dryer (Aeromatic AG, Germany). The inlet airflow was set at 50 m ³ /h and at ambient temperature.

For the abalone feed formulation, the centrifuged biomass was re-suspended in an artificial salt water (ASW) buffer. The re-suspended buffer was added to abalone feed and cold extruded using a screw extruder and dried in a convection oven (at 30°C) to a final moisture content of -10% m/m. The re-suspended biomass was extruded at a temperature in the range of 30 to 50°C. However, preferably the biomass paste is extruded at a temperature of 45°C. Results

A maximum cell concentration of 8.4x10 ⁹ cells/ml and optical density of 37.9 was obtained when Debaryomyces hansenii was cultivated in medium containing 25 g/l HTM and 24 g/l CSL after 22 hours of cultivation (Figure 8).

Airflow and temperature was maintained at 1 v/v/m and 24°C throughout the fermentation. pH was controlled automatically at 5.6 using 10% v/v H ₂ S0 ₄ or 25% v/v NH ₄ OH.PO ₂ was maintained above 30% by ramping up agitation speed when necessary. A maximum oxygen utilization rate (OUR) of 200 mMol/l/h was achieved in this fermentation (Figure 9).

The key responses measured were growth rate, cell concentration, cell productivity and cell yields on protein (YPP), sugar (YPS) and oxygen (YPO) (Figure 10).

A growth rate of 0.3/h was measured. A maximum cell concentration of 8.4x10 ⁰⁹ cells/ml and cell productivity of 3.8x10 ¹¹ cells/ml/h was obtained by cultivating Debaryomyces hansenii in the fermentation medium. Debaryomyces hansenii cell yields on protein, sugar and oxygen were 3.6x10 ¹¹ cells/g, 3.5x10 ¹¹ cells/g and 4.2x10 ¹¹ cells/g respectively (Figure 10).

The liquid product had a half-life of 381 days (storage at 4°C). Formulated to a final product initial count of 2x10 ¹⁰ CFU/ml, the shelf-life is 2535 days (to a final product count of 1x10 ⁸ CFU/ml).

The extruded product had a half-life of 27.5 days (storage at 4°C). Formulated to a final product initial count of 2x10 ¹⁰ CFU/ml, the shelf-life is 192.5 days (to a final product count of 1x10 ⁸ CFU/ml).

The Debaryomyces hansenii abalone feed has a half-life of 101.2 days (storage at 4°C). Formulated to a final product initial count of 2x10 ¹⁰ CFU/ml, the shelf-life is 708.4 days (to a final product count of 1x10 ⁸ CFU/ml). Production technology for Debaryomyces hansenii probiotics was previously unknown. There are no reports in the literature of actual yeast production according to parameters for commercial performance such as concentration, productivity and yield co-efficients of biomass or cell number. Papouskova and Sychrova (2007) suggested a cultivation temperature of 23°C and a pH of 6.5 for the production of xylitol (their objective was not to produce biomass). Other papers have mentioned an optimal temperature range of 28-30°C. The only precedent related to biomass production of this yeast was the description of growth from the studies of Macey & Coyne (2006). These studies described the use of standard YPD media, a temperature of 20°C and pH of 7.0.

The present invention describes a process for the commercial production of Debaryomyces hansenii using a proprietary yeast growth medium which improved cell productivity by 30% and reduced the cost of production by 42.7%.

Further, the findings of our research led us to the conclusion that the optimal temperature for the process described herein is 24°C, as opposed to what has previously been described. This new cultivation temperature resulted in a 449-fold improvement in cell productivity. By adjusting the pH to 5.6, a further 95% improvement in cell productivity was observed.

By using CSL as a nutrient source, at the optimum pH and temperature, the performance of the process was significantly improved and resulted in a surprising 176% increase in cell concentration, a 2382-fold increase in cell productivity and a 644% decrease in cost of production.

The combination of the abovementioned factors has led to a novel process for the commercial production of Debaryomyces hansenii as a probiotic coupled with significant increases in productivity, concentration and yield, together with a substantial reduction in the cost of production.

The cumulative effect of the process of this example has resulted in a 2075- fold increase in productivity of the previously known production process. This increased output may be attributed to the use of the new growth medium, together with an optimal temperature and pH. The productivity of the process was further improved by the use of CSL as a nutrient and this resulted in a further increase of 15% in cell productivity and a 66% lower material cost of production in comparison to the previous best process for producing Debaryomyces hansenii.

EXAMPLE 3

Comparative assessment of production media and culture conditions

A comparative assessment of the process of the present invention and the process described by Macey & Coyne (2006) was conducted. The inventors investigated the cost of production and the cell productivity of cultures that were grown according to the conditions and media set out in Macey & Coyne versus the production medium of the present invention.

Macey & Coyne cultured Vibrio midae in marine broth (MB), made up as follows: [(wt/vol) 3% NaCI, 0.23% MgCI ₂ .6H ₂ 0, 0.03% KCI, 0.2% glucose, 0.5% casamino acids, 0.1 % yeast extract] with shaking at 100 rpm at 22°C and maintained on marine agar (MA) [MB supplemented with 2% bacteriological agar (wt/vol), Unilab], The authors cultured Debaryomyces hansenii in yeast peptone D-Glucose (YPD) broth, made up as follows: [(wt/vol) 1 % yeast extract, 2% peptone, 2% D- glucose) and maintained on YPD agar [YPD broth supplemented with 1.5% bacteriological agar (wt/vol), Unilab].

Results

The results of the comparative assessment of the growth media and process of the present invention and the growth media and processes of the prior art are set out in Table 1.

The media and process described herein produce a Vibrio midae biomass concentration up to a maximum of 46.0 g/l and a biomass productivity of 11.4 g/l/h. Likewise the media and process described herein produce a Debaryomyces hansenii biomass concentration of up to a maximum of 133.7 g/l and a biomass productivity of 6.1 g/l/h. Table 1 : Comparative assessment of the productivity and cost of the production of abalone probiotics using the methods and media of the present invention and the methods and media described in Mace and Co ne 2006 ^{' '}

From the results in Table 1 it is apparent that the growth media and intensive production processes developed by the inventors for the production of Vibrio midae and Debaryomyces hansenii for use as probiotics in aquaculture have led to a significant cost decrease for commercially producing these microorganisms together with a corresponding increase in the productivity of the respective cultures.

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