CHAPTER 1 

Bioactive peptides derived from whey proteins

 

Pirkko Antila*, Anne Pihlanto-Leppälä^†  and Ilari Paakkari^‡

 

⁽KEYWORDS: FUNCTIONALITY, BIOLOGICAL, PEPTIDES, MILK)

 

* University of Helsinki, Department of Food Technology, Box 27, FIN-00014 Helsinki, Finland

^†Agricultural Research Centre of Finland, Food Research Institute, FIN-31600 Jokioinen, Finland

^‡ University of Helsinki,  Department of Pharmacology and Toxicology, BOX 81, FIN-00014 Helsinki Finland

Correspondence:

Pirkko Antila

Harjukatu 21 C

05900 Hyvinkää

Finland

Tel.+ 358-19-430104

Fax + 358-19-430104

ABSTRACT

 

The studies concerning the formation of bioactive peptides from whey proteins have been reviewed.

 

Digestion of β-lactoglobulin (β-lg) by different proteolytic enzymes releases  tetrapeptide β-lactorphin, β-lg f102-105 (Tyr-Leu-Leu-Phe) and β-lactotensin, β-lg f146-149, (His-Ile-Arg-Leu). The tetrapeptide α-lactorphin, α-la f50-53 (Tyr-Gly-Leu-Phe), can be produced during the proteolysis of α-lactalbumin (α-la) by pepsin. 

α-lactorphin have been shown to have a weak morphine like opioid property antagonized by naloxone. The same kind of pharmacological studies indicated that both β-lactorphin and β-lactotensin has a non opioid smooth muscle stimulatory effect not antagonized by naloxone.

 

The tetrapeptides of major whey proteins (β-lg, α-la) β-lactorphin, β-lactotensin and  α-lactorphin as well as β-lg f142-148 and albutensin A, serum albumin f208-216 have also shown to have an angiotensin-I-converting enzyme inhibitory effect connected with the antihypertensive influences.

 

The reviewed studies indicate that peptides released at least during in vitro proteolysis of whey proteins can have different bioactive properties. These observations confirm the value of whey proteins as source of nutraceutical compounds in human nutrition.

 

INTRODUCTION

 

Bioactive peptides have been identified as decomposition products of several food proteins (Brantl et al., 1979; Zioudrou et al., 1979; Loukas et al., 1983). Milk proteins are the most important sources of bioactive peptides. These have been shown to have various activities including opiate, antithrombotic or antihypertensive activity, immunomodulating or mineral utilization properties. Some of them have been known to influence in insulin secretion or the motility and secretion of the intestine (Daniel et al., 1990).

 

The most well known milk peptides are different casomorphins, the decomposition products of β-casein. As yet, only β-casomorphin-11 and α-casein phosphopeptide have been characterized as in vivo decomposition products of milk proteins (Meisel and Frister 1988; 1989).

 

In contrast to caseins, bioactive peptides obtained from whey proteins, and their physiological effects have been less studied and characterized. Yoshikawa et al. (1986) were the first ones who studied whey proteins in this respect. They synthesized tetrapeptides in the amide form on the basis of the opioid like fragments Tyr-X₁-X₁-Phe contained in the primary structures of α-la (both bovine and human) and β-lg (bovine). The fragment containing residue 50-53 of α-la (Tyr-Gly-Leu-Phe) in the amide form, was called α-lactorphin. Analogously, the 102-105 amide fragment of β-lg (Tyr-Leu-Leu-Phe) was called β-lactorphin (Figure 1). The activity of these synthetic peptides was low, both in receptor assay and pharmacodynamic measurements in guinea pig ileum and mouse was deferens preparations in vitro. Naloxone antagonized the effect of α-lactorphin completely in guinea pig ileum but affected the response of β-lactorphin only partially. Yamuchi (1992) has reported that two peptides derived from serum albumin (SA) and β-lg induced contraction of guinea pig ileum longitudinal muscle when test was done without electric stimulation in the absence of agonist. The peptides were called as “peptides acting on smooth muscle“ and they contained SA f208-216 (albutensin A) and β-lg f146-149 (β-lactotensin)

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α-la

41                                                                50                  53                                              60

Ile- Val-Gln-Asn-Asn-Asp-Ser-Thr-Glu-Tyr-Gly-Leu-Phe-Gln-Ile-Asn-Asn-Lys-Ile-Trp-

 

 

β-lg

101  102                     105                            110                                                                   120

Lys-Tyr-Leu-Leu-Phe-Cys-Met-Glu-Asn-Ser-Ala-Glu-Pro-Glu-Glu-Ser-Leu-Ala-Cys-Gln-

141                                  146                  149                                       160

Lys-Ala-Leu-Pro-Met-His-Ile-Arg-Leu-Ser-Phe-Asn-Pro-Thr-Gln-Leu-Glu-Glu-Gln-Cys-

 

 

SA

201                                               208                                                      216                     220

Ile-Gln-Lys-Phe-Gly-Glu-Arg-Ala-Leu-Lys-Ala-Trp-Ser-Val-Ala-Arg-Leu-Ser-Gln-Lys-

391                                                           399                             404                                    410

Asp-Gln-Phe-Glu-Lys-Leu-Gly-Glu-Tyr-Gly-Phe-Gln-Asn-Ala-Leu-Ile-Val-Arg-Tyr-Thr-

 

 

Bioactive peptides	Regions in the primary structure	Bioactivity
α-lactorphin	α-la f50-53	opioid agonist
Serorphin	SA f399-404	opioid agonist
β-lactorphin	β-lg f102-105	peptide acting on smooth muscle
β-lactotensin	β-lg f146-149	peptide acting on smooth muscle
albutensin A	SA f208-216	peptide acting on smooth muscle

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Figure 1. Bioactive peptide sequences in the primary structure of bovine α-la, β-lg and SA (Yoshikawa et al., 1986; Antila et al., 1991; Yamauchi, 1992).

 

 

THE FINNISH STUDIES CONCERNING BIOACTIVE PEPTIDES DERIVED FROM WHEY PROTEINS

 

The purpose of the first study (Antila et al., 1991) was to determine whether the opioid-like tetrapeptides α-lactorphin and β-lactorphin  described by Yoshikawa et al. (1986) were released from bovine α-la and β-lg using in vitro proteolysis and different proteolytic enzymes. The purpose of the second study (Pihlanto-Leppälä et al., 1997) was to determine  whether β-lactotensin was released from bovine β-lg by in vitro proteolysis  using different proteolytic enzymes. The pharmacological activity of these tetrapeptides was characterized in a receptor assay and in guinea pig ileum in vitro using synthetic fragments.

 

Materials and methods used are described in detail in the original articles. Peptides in hydrolysates were separated by reversed phase chromatography and identified by amino acid and sequence analysis. The results obtained during in vitro proteolysis  of β-lg and the release of β-lactorphin and β-lactotensin are presented in Table 1. The analogical results of _α-la and α-lactorphin are in Table 2.

 

As shown in Table 1. β-lg f102-105 and longer fragments containing this fragment, were released only in samples predigested with pepsin, when combined with proteolysis by trypsin, trypsin and chymotrypsin or pancreatin. β-lg f146-149 was released during hydrolysis with chymotrypsin. A longer fragment containing β-lg f146-149 or shorter fragments were found in hydrolyses with other enzymes. According to peak area after HPLC the amount of liberated and isolated  β-lg peptides were calculated to be: β-lactorphin 1.7 mg/g proteolyzed β-lg and β-lactotensin 4.0 mg/g proteolyzed β-lg. Synthetic peptides were used as standard. Chymotrypsin seems to be necessary for releasing the β-lactotensin from β-lg while pepsin and trypsin were needed for releasing of β-lactorphin.

 

It is known that β-lg is very resistant to proteolysis by gastric enzymes in the stomach (Savalle et al., 1988) or to proteolysis by pepsin in vitro (Reddy et al., 1988). On the other hand it has been established that pepsin makes the molecular structure of β-lg more susceptible to proteolysis by other proteinases ( Porter et al., 1984; Rothenbuler and Kinsella, 1985; Antila, 1988). As well as pepsin, heat treatments also increases the susceptibility of β-lg to proteolysis (Reddy et al., 1988; Antila, 1988). Results obtained in our studies showed that proteolysis caused by pepsin and trypsin was considerable greater in heated β-lg than in native β-lg. β-lactorphin was also released during the proteolysis of heated β-lg.

 

In vitro proteolysis of α-la released α-lactorphin during proteolysis by pepsin alone, pepsin and trypsin or pepsin, trypsin and chymotrypsin as shown on Table 2. According to peak area after HPLC the amount of liberated and isolated α-lactorphin  was calculated to be 5.0 mg/g proteolyzed α-la. Synthetic α-lactorphin was used as standard.

 

Results of the pharmacological activity of synthetic α- and β-lactorphins are presented in Table 3. The effects in  guinea pig ileum of α- and β-lactorphin were apparent at concentration of 10^-4 M in contrast to morphine that inhibited contractions at 10^-6 M. The results indicated that α-lactorphin exerts a naloxone sensitive inhibition of the smooth muscle contractions similar to that of morphine. In contrast, β-lactorphin induced a stimulation of the smooth muscle, that was not sensitive to naloxone. The affinity of α-lactorphin to opioid receptors was about 1000-fold lower than that of morphine. Binding of β-lactorphin to the opioid receptors was similar as that of α-lactorphin. According to the results it was concluded that α-lactorphin exerted a weak but consistent opioid property in the smooth muscle and receptor binding while β-lactorphin in spite of the similar receptor binding affinity exerted an apparently non-opioid stimulatory effect on the guinea pig ileum. The detailed data of the pharmacological activity of the synthetic α- and β-lactorphins are presented in the study of Paakkari et al. (1994).

 

In the pharmacological studies of β-lactotensin (Pihlanto-Leppälä et al., 1997). morphine inhibited the contractions of coaxially stimulated guinea pig ileum at concentrations of 10^-8-10^-5 M. The effect of morphine was completely antagonized by naloxone (10^-6 M), β-lactotensin at concentrations 10^-8-10^-6 M gave no reaction while at 10^-5-10^-4 M a contractile response was observed. The effect of ß-lactotensin was opposite to morphine which was used as reference. Moreover, the opioid antagonist naloxone (10^-6 M) did not inhibit the effect of ß-lactotensin. The stimulatory effect of ß-lactotensin on smooth muscle was similar to ß-lactorphin (Antila et al., 1991). The results indicate that smooth muscle contracting effect of ß-lactotensin as well as ß-lactorphin was not mediated by an opioid mechanism and thus remains unclear.

 

OTHER STUDIES CONCERNING BIOACTIVE PEPTIDES OF WHEY PROTEINS AND THEIR PHYSIOLOGICAL EFFECTS

 

Novel angiotensin-I-converting enzyme (ACE)  inhibitory activities were detected in synthetic peptides corresponding the sequences of β-lg and α-la. ACE is part of the rennin-angiotensin system, which has been implicated in blood pressure regulation and hypertension. Rennin acts on angiotensinogen and releases a largely inactive angiotensin I which is then converted to the active peptide hormone  angiotensin II by ACE. The tetrapeptides α-lactorphin, β-lactorphin and β-lactotensin and related peptides were shown to have ACE-inhibitory activity (Mullally et al., 1996). using hippuryl-histidyl-leucine as substrate inhibit ACE activity in the following order: β-lactorphin  (IC₅₀ = 171.8 μM) > α-lactorphin (IC₅₀ = 733.3 μM) > β-lactotensin (IC₅₀ = 1153.2 μM). The N-terminal dipeptide of β-lactorphin was  found to be the most potent inhibitor (IC₅₀ = 122.1 μM). β-lg peptide obtained after tryptic digest of β-lg and identified as β-lg f142-146 (IC₅₀ = 42.6 μM) was found to be the most active ACE-inhibitory whey peptide so far reported (Mullally et al., 1997).

 

Several casein-derived ACE-inhibitory peptides having also other biological activities have been reported e.g. casomorphin-7 (Meisel and Schlimme 1994). Chiba and Yoshikawa (1991) characterized a multifunctional bioactive peptide, albutensin A, serum albumin f208-216.

CONCLUSIONS

 

According to the reviewed studies α- and β-lactorphins and β-lactotensin are all multifunctional peptides derived from whey proteins. Using different proteolytic enzymes they can be released at least during in vitro proteolytic conditions which in certain degree resemble the conditions during in vivo digestion. Pepsin in the stomach can convert whey proteins, especially β-lg, more susceptible to the proteolytic enzymes in the small intestine. In processed milk products, in particular heat treatment can increase susceptibility of whey proteins to the proteolysis both in vitro and in vivo.

 

If these kind of bioactive, nutraceutical, peptides are available in the human organisms how well they can be utilized and how effective their biological properties can be. All these kind of questions will need further elucidation.

 

Table 1. Proteolysis of β-lg by different enzymes and detection by HPLC of β-lg  peptides 102-105 (Tyr-Leu-Leu-Phe) and 146-149 (His-Ile-Arg-Leu) in the fraction soluble in trichloroacetic acid (Antila et al., 1991; Pihlanto-Leppälä et al., 1997).

 

Substrate	Enzymesa	Duration (h) of proteolysis at 37 °C	β-lg fragment
β-lg 0.3 % native	trypsin	3	142-148
	trypsin	24	146-148
	chymotrypsin	3	146-149
	chymotrypsin	24	146-149
	pepsin and trypsin	3 24	102-105 146-148
	pepsin + chymotrypsin	3 24	146-149
	pepsin + chymotrypsin and trypsin	3 24	102-105 146-148
	pepsin + pancreatin	3 24	101-112
β-lg 0.3 % heated for   1 h at  80 °C,  pH 8.0	pepsin + trypsin	3 24	102-105

a) Enzyme/Protein ratio 1:200 in all experiments

 

 

 

Table 2. Proteolysis of α-la by different enzymes and detection by HPLC of α-la peptide 50-53 (Tyr-Gly-Leu-Phe) in the fraction soluble in trichloroacetic acid (Antila et al., 1991)

substrate	Enzymesa	Duration (h) of proteolysis at 37 °C	α-la fragment
α-la 0.3 % native	pepsin	3	50-53
	pepsin + trypsin	3 24	50-53
	pepsin + trypsin and chymotrypsin	3 24	50-54

a) Enzyme/Protein ratio 1:200 in all experiments