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Report No. 006 Development of a functional collagen hydrolysate using ginger enzyme
Report No. Development of a functional collagen hydrolysate using ginger enzyme
006

Overview

Collagen hydrolysate, also known as gelatin hydrolysate or collagen peptide, is produced by enzymatic partial hydrolysis of collagen thermally extracted from skin, bone, and fish scales. Many studies have reported beneficial effects of oral intake of collagen hydrolysate on various tissues, including bone, joints, skin, and blood vessels. Although the mechanism of these effects of collagen hydrolysate has not been elucidated, it has recently been demonstrated that dipeptides and tripeptides*1 containing hydroxyproline (Hyp; a modified amino acid specific to collagen) are absorbed into the blood at significantly high concentrations after oral ingestion of collagen hydrolysate (Reference 1). The bioactivity and absorption/metabolism of Hyp-containing oligopeptides are currently under intense investigation.
It is known that protease is present in ginger rhizome from 50 years ago, and its meat tenderizing effect has been studied for a long time. Ginger is used for cooking meat, not only to add flavor but also to tenderize the meat by the enzyme. We have conducted research focusing on the strong protease activity of ginger, and successfully developed a novel collagen hydrolysate using ginger enzyme. This report introduces the ginger-degraded collagen hydrolysate (GDCH), and the function of Hyp-containing peptides specifically contained in GDCH.
*1: A chain of several to dozens of amino acids connected is called peptide. The “dipeptide” consists of two amino acids, and the “tripeptide” consists of three amino acids. Protein ingested as a meal is digested into peptides and further into amino acids, but some parts are absorbed as dipeptides and tripeptides that perform their function in the body.

Development of a novel collagen hydrolysate using ginger enzyme.

By reacting ginger powder prepared by clash treatment followed by drying, heat-denatured collagen (gelatin) is efficiently digested to peptides and reduced in molecular weight by the effect of ginger enzyme (Figure 1). Analysis of the collagen hydrolysate prepared by this reaction showed that a substantial number of X-Hyp-Gly-type tripeptides*2 was generated (Reference 2). X-Hyp-Gly-type tripeptides are not contained in existing collagen hydrolysate products, indicating that this type of tripeptide is unique to GDCH (Figure 2).
*2: The “X-Hyp-Gly” is a tripeptide consisting of an amino acid X, Hyp, and Gly in sequence. There are multiple X-Hyp-Gly-type tripeptides because X can be a variety of amino acids.

Figure 1. Digestion of collagen using ginger enzyme.

Figure 2. Content of X-Hyp-Gly-type tripeptides in various collagen hydrolysates.

We compared blood absorption of X-Hyp-Gly-type tripeptides between GDCH and collagen hydrolysate prepared with other enzymes (control) by oral administration of the test samples to mice. The result showed that the concentration of X-Hyp-Gly-type tripeptides in blood was significantly higher in mice administered GDCH (Figure 3). This result indicated that at least some of the X-Hyp-Gly-type tripeptides contained in GDCH were absorbed into the blood without being degraded in the gastrointestinal tract. The X-Hyp-Gly-type tripeptides transported into the blood might exert various functions after delivery to tissues through the bloodstream.
In the following chapters, we briefly introduce physiological functions of X-Hyp-Gly-type tripeptides as revealed by our research.


Figure 3. Maximum concentration of X-Hyp-Gly-type tripeptides in blood after oral administration of GDCH. *p < 0.05, **p < 0.01.

Promotion of osteoblast differentiation by X-Hyp-Gly-type tripeptides

In exploring physiological function of X-Hyp-Gly-type tripeptides, we found that this type of peptide promotes differentiation of bone-forming cells, osteoblasts (Reference 3). By culturing mouse-derived osteoblasts with addition of X-Hyp-Gly-type tripeptides, osteoblast differentiation markers, including alkaline phosphatase (ALP) activity and mineralization*3, were significantly increased (Figure 4). In addition, enhancement of collagen secretion was observed by Ala-Hyp-Gly and Leu-Hyp-Gly at an early stage of differentiation. These promoting activities of X-Hyp-Gly-type tripeptides on osteoblast differentiation suggest that ingestion of GDCH containing those peptides has beneficial effects on bone.
*3: Osteoblasts build bone by secreting collagen and depositing calcium phosphate around themselves, which is called “mineralization”.

Figure 4. promotional effects of X-Hyp-Gly-type tripeptides on osteoblast differentiation. *p < 0.05, **p < 0.01.

Angiotensin converting enzyme inhibitory activity of X-Hyp-Gly-type tripeptides

As one of the physiological functions of X-Hyp-Gly-type tripeptides, we found angiotensin converting enzyme (ACE)*4 inhibitory activity leading to blood pressure reduction (Reference 4). A variety of X-Hyp-Gly-type tripeptides showed ACE inhibitory activity, and it was demonstrated that substitution of Hyp with proline (Pro) significantly reduced the inhibitory activity (Figure 5). Approximately half of Pro, a major collagen-constituting amino acid, is converted to Hyp by hydroxylation. Our data indicate that this collagen-specific modification largely enhance the ACE inhibitory activity of peptides. Since X-Hyp-Gly-type tripeptides are absorbed into the blood at high concentrations after ingestion of GDCH, we can expect blood pressure lowering effects of GDCH.
*4: ACE converts an inactive peptide angiotensin I into a potent vasoconstrictor angiotensin II, indicating that inhibition of ACE activity results in suppression of elevated blood pressure.

Figure 5. ACE inhibitory activity of peptides.
50% inhibitory concentration: the lower the value, the stronger the inhibitory activity of the peptide.

Conclusion

The newly developed collagen hydrolysate GDCH contains unique X-Hyp-Gly-type tripeptides, and they have various biological functions. We also found that collagen-specific cyclic dipeptides, cyclo(X-Hyp), are generated from X-Hyp-Gly-type tripeptides (Reference 5). Due to their unique structure, cyclo(X-Hyp) may also have physiological function. We will continue to investigate the health benefits of GDCH.

References

1. Iwai K, Hasegawa T, Taguchi Y, Morimatsu F, Sato K, Nakamura Y, Higashi A, Kido Y, Nakabo Y, Ohtsuki K. Identification of food-derived collagen peptides in human blood after oral ingestion of gelatin hydrolysates. J Agric Food Chem. 53, 6531-6536 (2005)
2. Taga Y, Kusubata M, Ogawa-Goto K, Hattori S. Efficient absorption of X-hydroxyproline (Hyp)-Gly after oral administration of a novel gelatin hydrolysate prepared using ginger protease. J Agric Food Chem. 64, 2962-2970 (2016)
3. Taga Y, Kusubata M, Ogawa-Goto K, Hattori S, Funato N. Collagen-derived X-Hyp-Gly-type tripeptides promote differentiation of MC3T3-E1 pre-osteoblasts. J Funct Foods. 46, 456-462 (2018)
4. Taga Y, Hayashida O, Ashour A, Amen Y, Kusubata M, Ogawa-Goto K, Shimizu K, Hattori S. Characterization of angiotensin-converting enzyme inhibitory activity of X-Hyp-Gly-type tripeptides: importance of collagen-specific prolyl hydroxylation. J Agric Food Chem. 66, 8737-8743 (2018)
5. Taga Y, Kusubata M, Ogawa-Goto K, Hattori S. Identification of collagen-derived hydroxyproline (Hyp)-containing cyclic dipeptides with high oral bioavailability: efficient formation of cyclo(X-Hyp) from X-Hyp-Gly-type tripeptides by heating. J Agric Food Chem. 65, 9514-9521 (2017)

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