The Bac-in-a-Box^® technology developed by Austrianova is a unique, novel and patented protective technology for aerobic and anaerobic living bacteria and yeast such as probiotics/microbiota that shields against

(i) the harmful effects of exposure to stomach acid and bile including protection from pH 2 for over 4 hours.
(ii) spoilage during storage, allowing storage at room temperature for over a year without significant loss of viability.

Austrianova is a Business-to-Business (B2B) company that offers its Bac-in-a-Box technology to improve the storage and delivery of living cell products (Koe, 2021). The advantages afforded by Bac-in-a-Box allow efficient delivery of lower doses of bacteria and yeast thereby enhancing safety and cost effectivity. This makes the technology ideal for the delivery of microbiota for the treatment of any disease arising as a result of dysbiosis where the bacterial species composition of the gut becomes abnormal (Aroila et al., 2023).

The material that comprises Bac-in-a-Box technology is a proprietary formulation of a cellulose derived polymer, thus it is naturally occurring, bio-sustainable and biocompatible and is “generally regarded as safe”. It can be sustainably sourced from plants or agricultural waste and is chemically modified using a proprietary method allowing it to become the ideal bioprotective material for encapsulation.

Unlike classical protective methods (such as spray drying), our cellulose sulphate formulation engulfs the probiotics so that they are entrapped within tiny porous beads. Bacteria or yeast from a small seed culture are resuspended in liquid cellulose sulphate and droplets containing the microorganisms are created that harden to jelly-bean like spherical microbeads or micro-capsules (Figure 1). Millions of these micro-capsules are produced in one production run that is performed at ambient temperature. Another novel aspect of the Bac-in-a-Box technology is that the bacteria or yeast are then grown or expanded within the micro-capsules (fermentation) and then freeze dried using a proprietary method so that they are in a “suspended animation” like state and can be stored at room temperature for a year or more (Figure 2). Bac-in-a-Box is thus very different to current methods used to protect bacteria and yeast where huge bio-fermenters are used to produce large amounts of the bacteria/ yeast which are then spray dried.

Figure 1. A schematic of the Bac-in-a-Box^® manufacturing process. (a) Living cells mixed with proprietary cellulose sulphate (CS) polymer are injected through a nozzle vibrating at high frequency. The jet of microbead-sized droplets then falls into a second polymer which (b) causes a porous membrane to form on the surface of the final microbead. (c) an electron micrograph of a single microbead. (d) a freeze-thraw fracture micrograph showing a side view of the gelated membrane and cells inside surrounded by cellulose. 

 

Protection from low pH in the stomach
The stomach is an acidic environment with the low pH in the stomach being lethal for most bacteria and yeasts. Bac-in-a-Box encapsulated bacteria, and also yeast, survive stomach acid at pH 2 for at least 4 hours (240 mins) and also survive subsequent addition of bile juice which is similarly highly bacteriocidal (Gunzburg et al., 2020).

• The acid protection afforded by Bac-in-a-Box is 4-5 logs better than the best competing  technologies (Salmons et al., 2020)
• The bacteria are later released from the capsules during transit through the intestines (Gunzburg et al., 2020).

This data has been independently confirmed and verified by a number of Austrianova’s partner companies including:

• one partner using Bac-in-a-Box to micro-encapsulate a well-known probiotic, who has shown efficient delivery and release of the bacteria in the gastric tract of humans.
• another partner using Bac-in-a-Box to efficiently deliver strictly anaerobic bacteria to the rumen of farm animals allowing rapid modification of the rumen microbiome.

Modifications made to the encapsulation parameters can result in micro-capsules that do not release the entrapped probiotic bacteria/yeast but instead release the proteins that they produce which escape via the maze like pores on the surface of the micro-capsule structure.

Long-term storage without refrigeration
Bac-in-a-Box micro-encapsulated bacteria/yeast can be kept at room temperature for storage and transportation and without needing a cool chain without appreciable loss in viability. Austrianova has shown good viability of various strains of micro-encapsulated aerobic and anaerobic bacteria and yeast after storage for over a year at room temperature (Dangerfield et al., 2018).

Areas of application other than the gastric tract
Austrianova’s proprietary encapsulation material, Gel8, that is used to produce Bac-in-a-Box has been approved as a cosmetic ingredient name by the International Nomenclature of Cosmetics Ingredients (INCI) and is listed in the INCIpedia. This opened the way to the company winning the L’Oreal “Innovation Runway” start-up challenge in 2017 (Teh, 2018) and an ongoing relationship with L’Oréal on a multi-pronged project to develop uses of Austrianova’s Bac-in-a-Box technology for innovative skin health/cosmetic products (Manalastas, 2019). Bac-in-a-Box also can be used in products such as soaps to balance the skin environment pH level (Tech Collective, 2019). There are also a number of non-medical, non-health, commercial uses of the Bac-in-a-Box® technology currently under development.

Packaging
Bac-in-a-Box can be formulated into bags, sticks, vials or caplets for use individually or as a “topping” on animal food/feed. Moreover, by mixing different batches of Bac-in-a-Box micro-capsules, each containing different bacterial stains, specific customised blends of bacteria can be created, for instance to alter the microbiome in the gut (Salmons and Gunzburg, 2019). Stick packaging of Bac-in-a-Box is particularly suitable when formulating for young children, older adults or patients who have difficulties in swallowing, since the tiny grain like size of the particles are easily consumed either directly or mixed in liquid drinks.

Summary
In summary, the broadly applicable Bac-in-a-Box technology can be used across many areas of application. Delivery to the gastric tract of humans, pets, farm and aquatic animals allows replacing or rebalancing the microbiome after antibiotic use, to correct dysbiosis and to improve general health status and restore optimal digestion. It can also facilitate the use of live oral vaccines or the production of recombinant proteins in a similar fashion. Bac-in-a-Box can also be applied to restore, replenish and regenerate the skin to maintain and promote skin health but also in wound healing. It can also be used as a means to retrieve bacteria and yeast from bioreactors as well as having other uses in agriculture, bioremediation and as bioindicators.

Visit Austrianova for more or use the Request Information to email the company directly.

References
Carlo Airola, Andrea Severino, Serena Porcari, William Fusco, Benjamin H. Mullish, Antonio Gasbarrini, Giovanni Cammarota, Francesca Romana Ponziani, Gianluca Ianiro. Future Modulation of Gut Microbiota: From Eubiotics to FMT, Engineered Bacteria, and Phage Therapy. Antibiotics 2023, 12, 868.

Tingmin Koe 27 July 2021. Against the number game: Singapore biotech firm receives seven-figure funding to grow probiotics encapsulation business. NutraIngredients Asia.

Walter H. Gunzburg, Myo Myint Aung, Pauline Toa, Shirelle Ng, Eliot Read, Wee Jin Tan, Lilli Brandtner, John Dangerfield, Brian Salmons. 2020. Efficient Protection of Microorganisms for Delivery to the Intestinal tract by Cellulose Sulphate Encapsulation. Microbial Cell Factories 19, 216. doi: 10.1186/s12934-020-01465-3.

Brian Salmons, John Dangerfield, Walter H. Gunzburg. 2020. Delivery of probiotics efficiently to the intestine: the acid test. NuFFooDS Spectrum Asia.

Brian Salmons, Walter H. Gunzburg. 2019. Treating diseases by redressing the microbiome balance. BioSpectrum Asia.

John A. Dangerfield, Elliot Read, Brian Salmons, Walter H. Gunzburg, W.H. (2018) A game-changing live cell encapsulation technology for enabling allogenic cell therapies and delivery of probiotics to the gut. Regenerative Research 7(1), 40

INCIpedia https://incipedia.personalcarecouncil.org

Annie Teh, 26 Jul 2018. 3 ways tech is changing the beauty industry. TechInAsia 

Alfonso Manalastas, 8 August 2019. Leaving a beauty mark in Asia’s growing urbanisation. e27

Tech Collective, 29 January 2019. We explore beauty technology in Southeast Asia. Tech Collective.