About P H B
for pet therapy
Polyhydroxybutyrate (PHB) made from a bacteria which originally live in the sea and salt lake. From a chemical point of view, PHB is a simple compound, a polymer of ketone bodies. PHB is known as an energy substrate for hunger saved in the bacteria and has been regarded as a candidate of biodegradable plastic. However, because it is too fragile to be used as a substrate of tissue engineering, it has not been realized as a bioplastic up to the present time. In contrast, we have been stacked to use of this compound in another biological field.
Since this is hydrolyzed and primarily used as an energy substrate in the enterobacteria, it may affect and modulate the composition of intestinal microbial flora.
This is a kind of “New-Prebiotics”.
Beyond Science Ltd. has been aiming at the industrialization of this compound as an effector of prebiotics. This is the straightforward way for realization of “Eatable Ketone body” according to social demand for pet health.
And we believe that this method is a useful tool to practice easy keto diet and ketogenic life for dogs, cats and so on. Our commitment is to be “beyond ketogenic”.
KtB-powder metabolized by enterobacteria
PHB is a beta-hydroxybutyrate (ketone body)-polymerized compound, in which ketone body connects with each other by an ester bond(1-2). Because mammalian enzymes cannot digest PHB , PHB goes through small intestine and reaches large intestine. Just enterobacteria can digest this compound by their own enzymes(2-3). As a result, enterobacteria produce ketone body in their bodies and metabolize it through a chain of chemical reactions for energy production. Thus, various enterobacteria, including good ones, are activated to growth. Thus, PHB can significantly improve microbiological environment of large intestine and this is the physiological basis of health benefits of PHB . The activation of enterobacteria through ketone body by PHB and the improvement of the environment of large intestine can induce various health benefits and we call these mechanisms as “New-Prebiotics”, which is a novel mode of “Prebiotics”. In addition, the ketone body can be diffused into the space of large intestine as well as consumption inside bacterial body. Epithelial cells of large intestine can absorb ketone body.
Obruca S, Sedlacek P, Krzyzanek V, et al. Accumulation of Poly(3-hydroxybutyrate) Helps Bacterial Cells to Survive Freezing. PLoS One. 2016;11(6):e0157778. Published 2016 Jun 17. doi:10.1371/journal.pone.0157778
Gowda U S V, Shivakumar S. Poly(-β-hydroxybutyrate) (PHB) depolymerase PHAZ Pen from Penicillium expansum: purification, characterization and kinetic studies. 3 Biotech. 2015;5(6):901–909. doi:10.1007/s13205-015-0287-4
Tseng CL, Chen HJ, Shaw GC. Identification and characterization of the Bacillus thuringiensis phaZ gene, encoding new intracellular poly-3-hydroxybutyrate depolymerase. J Bacteriol. 2006;188(21):7592–7599. doi:10.1128/JB.00729-06
Polyhydroxybutyrate (PHB) as a main component of KtB-powder
Various bacteria can save polyhydroxybutyrate (PHB) into granule-like shaped body inside the bacteria for energy substate(4). This is supposed to be a sort of bacterial insurance against their food crisis. Beyond Science Ltd. makes use of a peculiar strain, in which PHB often occupies over 80% of all space of the bacterial body. In this bacterial culture, PHB can be refined as pure KtB-powder by a simple method (Patent: WO2019/035486) (5-6). Since PHB is almost tasteless and odorless, this is one of the best ingredients of pet food for dogs and cats.
4. Wang X, Jiang XR, Wu F, Ma Y, Che X, Chen X, Liu P, Zhang W, Ma X, Chen GQ. Microbial Poly-3-Hydroxybutyrate (PHB) as a Feed Additive for Fishes and Piglets. Biotechnol J. 2019 Dec;14(12):e1900132. doi: 10.1002/biot.201900132. Epub 2019 Jun 27.
5. Yu LP, Yan X, Zhang X, Chen XB, Wu Q, Jiang XR, Chen GQ. Biosynthesis of functional polyhydroxyalkanoates by engineered Halomonas bluephagenesis. Metab Eng. 2020 May;59:119-130. doi: 10.1016/j.ymben.2020.02.005. Epub 2020 Feb 29.
6. Yokaryo H, Teruya M, Hanashiro R, Goda M, Tokiwa Y. Direct Production of (R)-3-Hydroxybutyric Acid of High Optical Purity by Halomonas sp. OITC1261 Under Aerobic conditions. Biotechnol J. 2018 Feb;13(2). doi: 10.1002/biot.201700343. Epub 2017 Oct 30.
P H B
Activation of regulatory T cells(7)
PHB makes enterobacteria produce ketone body inside bacterial body and activates the growth of various enterobacteria through energy production due to increase in ketone body. Good enterobacteria are supposed to activate regulatory T cells and suppress unnecessary immunological reactions(8-9). Because PHB induces the increase in good enterobacteria, regulatory T cells are activated through macrophages of Payer board of the large intestine. Regulatory T cells can suppress excess symptoms of various inflammations such as allergy and autoimmunity and return to the basal line of the reaction.
7. Sakaguchi S, Mikami N, Wing JB, Tanaka A, Ichiyama K, Ohkura N. Regulatory T Cells and Human Disease. Annu Rev Immunol. 2020 Feb 4. doi: 10.1146/annurev-immunol-042718-041717.
8. Ladinsky MS, Araujo LP, Zhang X, Veltri J, Galan-Diez M, Soualhi S, Lee C, Irie K, Pinker EY, Narushima S, Bandyopadhyay S, Nagayama M, Elhenawy W, Coombes BK, Ferraris RP, Honda K, Iliev ID, Gao N, Bjorkman PJ, Ivanov II. Endocytosis of commensal antigens by intestinal epithelial cells regulates mucosal T cell homeostasis. Science. 2019 Mar 8;363(6431). pii: eaat4042. doi: 10.1126/science.aat4042.
9. Narushima S, Sugiura Y, Oshima K, Atarashi K, Hattori M, Suematsu M, Honda K. Characterization of the 17 strains of regulatory T cell-inducing human-derived Clostridia. Gut Microbes. 2014 May-Jun;5(3):333-9. doi: 10.4161/gmic.28572. Epub 2014 Mar 18.
Sustained increase in ketone body by PHB (10)
PHB can induce the sustained increase the concentrations of blood ketone body, which is in a sharp contrast to that of ketone ester, another type of ketone donor. Ketone ester can rapidly increase the concentrations of ketone body within several minutes but returned to the baseline within 5 hours(11-12). In contrast, PHB needs 5 hours to begin the increase, but induces the sustained increase at least over 10 hours. This may be due to digestion just by enterobacteria, but not to digestive enzymes of small intestine. Further, this sustained increase in ketone body is the biological key to the preventive effects against various lifestyle-related diseases.
10. Patent number: WO2019/035486
11. Abdul Kadir A, Clarke K, Evans RD. Cardiac ketone body metabolism. Biochim Biophys Acta Mol Basis Dis. 2020 Jun 1;1866(6):165739. doi: 10.1016/j.bbadis.2020.165739. Epub 2020 Feb 19
12. Newman JC, Verdin E. Ketone bodies as signaling metabolites. Trends Endocrinol Metab. 2014;25(1):42–52. doi:10.1016/j.tem.2013.09.002
P H B
A novel type of Prebiotics
The first biological target of PHB may be enterobacteria, already suggested by a prior art (patent: WO2005/021013). Pigs, kept for 5 days or 4 weeks and fed with PHB5%, increased the amount of excrement and defecation frequency(13). These results suggest that the number of enterobacteria is increased. In addition, PHB significantly decreased the amount of odor ingredients. Previous reports suggested that PHB can be digested by enzymes of enterobacteria. Thus, PHB can increase the ketone body and activate the growth of the bacteria.
P H B
13. Patent number: WO2005/021013
Ketone body as a promising molecule of anti-aging
Ketone body, which produces energy within mitochondria of the cells, is a simple C4 organic acid composed of carboxylic acid and hydroxyl group and thus two ketone body can form a connection through an ester bond. When great number of ketone body exists, they can form a polymer, PHB. Ketone body is no longer regarded as a metabolic intermediate, as it regulates a broad range of physiological events at cellular and systemic levels. Importantly, ketone body functions as a stress response to maintain redox homeostasis in response to environmental and metabolic challenges(14-15).
14. Veech RL, Bradshaw PC, Clarke K, Curtis W, Pawlosky R, King MT.Ketone bodies mimic the life span extending properties of caloric restriction. IUBMB Life. 2017 May;69(5):305-314. doi: 10.1002/iub.1627. Epub 2017 Apr 3.
15. Roberts MN, Wallace MA, Tomilov AA, Zhou Z, Marcotte GR, Tran D, Perez G, Gutierrez-Casado E, Koike S, Knotts TA, Imai DM, Griffey SM, Kim K, Hagopian K, McMackin MZ, Haj FG, Baar K, Cortopassi GA, Ramsey JJ, Lopez-Dominguez JA. A Ketogenic Diet Extends Longevity and Healthspan in Adult Mice. Cell Metab. 2017 Sep 5;26(3):539-546.e5. doi: 10.1016/j.cmet.2017.08.005. Erratum in: Cell Metab. 2018 May 1;27(5):1156.
Three types of ketone donor
ketone donor is defined as substance which produces ketone body by enzymes of the small and large intestine. The most prominent feature is the production of ketone body under no influence of insulin. Up to date, there are three types of ketone donors. One is ketone body salt, for example, sodium salt of ketone body(16-17). The second is ketone ester, which ketone body is connected to alcohol through an ester bond(11-12). The last is PHB, which is a polymer of ketone body(10). I have focused on PHB as a mammals’ supplement for these three years. Ketone body and ketone ester is hydrolyzed by enzymes of the small intestine and PHB is hydrolyzed by those of the large intestine. This difference makes a big difference of physiological effects by them in the point that just PHB can induce sustained increase in ketone body concentration.
16. Patent number: WO2015/072456
17. Patents number: WO2008/120778