Safety Assessment of Bovine Milk Proteins and Protein Derivatives as Used in Cosmetics.

The Expert Panel for Cosmetic Ingredient Safety (Panel) reviewed the safety of 16 bovine milk proteins and protein-derived ingredients, which function mainly as skin and hair conditioning agents in personal care products. The Panel reviewed relevant data provided in this safety assessment, and concluded that these ingredients are safe in the present practices of use and concentration.

The structure and properties of MFG-E8 and the In vitro assessment of its toxic effects on myoblast cells.

Four high-molecular-weight protein fractions of milk fat globule membrane (MFGM) were isolated from bovine milk. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), MALDI-TOF/TOF™ and Liquid chromatography electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) were used to measure the molecular sizes of the MFGM. Fourier transform infrared spectroscopy (FT-IR) and circular dichroism (CD) were performed to determine the conformations of the MFGM. The results showed that the main protein (98.33%) in MFGM protein fraction 2 was Milk fat globule epidermal growth factor-VIII (MFG-E8), with a molecular weight of 47.82 kDa. The secondary structural component measurements showed that the MFG-E8 consisted of 5% helix, 70% sheet and 25% random coil, and the results matched well with the prediction by SSPro 5.1 bioinformatic analysis. The thermograms analysis revealed that Td and△H of MFG-E8 were 60.50°Cand 132.29 kJ/mol. The in vitro digestibility of MFG-E8 showed that it can be enzymatically hydrolyzed in the stomach and relatively stable in the intestinal fluid. The in vitro C2C12 and Caco2 cell activity tests indicated that MFG-E8 promoted the proliferation of C2C12 myoblast cells without cytotoxicity. The biological functional properties of MFG-E8 may be related to the fact that MFG-E8 possesses a high level of ß-sheet structure. Our results suggested that MFG-E8 possesses broad prospects not only for use in functional food products but also as a source of natural anti-sarcopenia drugs.

Endothelial Immune Activation by Medin: Potential Role in Cerebrovascular Disease and Reversal by Monosialoganglioside-Containing Nanoliposomes.

Background The function of medin, one of the most common human amyloid proteins that accumulates in the vasculature with aging, remains unknown. We aim to probe medin's role in cerebrovascular disease by comparing cerebral arterial medin content between cognitively normal and vascular dementia (VaD) patients and studying its effects on endothelial cell (EC) immune activation and neuroinflammation. We also tested whether monosialoganglioside-containing nanoliposomes could reverse medin's adverse effects. Methods and Results Cerebral artery medin and astrocyte activation were measured and compared between VaD and cognitively normal elderly brain donors. ECs were exposed to physiologic dose of medin (5 µmol/L), and viability and immune activation (interleukin-8, interleukin-6, intercellular adhesion molecule-1, and plasminogen activator inhibitor-1) were measured without or with monosialoganglioside-containing nanoliposomes (300 µg/mL). Astrocytes were exposed to vehicle, medin, medin-treated ECs, or their conditioned media, and interleukin-8 production was compared. Cerebral collateral arterial and parenchymal arteriole medin, white matter lesion scores, and astrocyte activation were higher in VaD versus cognitively normal donors. Medin induced EC immune activation (increased interleukin-8, interleukin-6, intercellular adhesion molecule-1, and plasminogen activator inhibitor-1) and reduced EC viability, which were reversed by monosialoganglioside-containing nanoliposomes. Interleukin-8 production was augmented when astrocytes were exposed to medin-treated ECs or their conditioned media. Conclusions Cerebral arterial medin is higher in VaD compared with cognitively normal patients. Medin induces EC immune activation that modulates astrocyte activation, and its effects are reversed by monosialoganglioside-containing nanoliposomes. Medin is a candidate novel risk factor for aging-related cerebrovascular disease and VaD.

Safety evaluation of a milk-based protein powder produced by a novel manufacturing technique.

TruActive™ NF is a novel, fat-free, milk-based protein powder to be added to food to increase protein content and is manufactured using non-thermal treatment to reduce potential pathogens most relevant to protecting public health. TruActive™ NF was evaluated for potential pathogens of concern to public health regulators; none were detected. The estimated daily intake (EDI) of TruActive™ NF at a 90th percentile consumption for the powder in nutritional beverages and bars is 14,700 mg/day. In vitro genotoxicity testing revealed that concentrations of TruActive™ NF up to 5000 µg/plate did not induce point mutations in selected strains. Oral administration of TruActive™ NF to male Sprague-Dawley rats in an in vivo mammalian chromosomal aberration assay did not induce chromosomal aberrations or significantly affect mitosis in bone marrow cells at 2000 mg/kg. Male and female Sprague-Dawley rats were administered TruActive™ NF at concentrations of 7.5%, 15%, and 30% of the diet during a 28-day subacute dietary study followed by a 14-day recovery period. Some parameters were altered at the 30% diet concentration. The No Observed Adverse Effect Level (NOAEL) in the 28-day dietary study was at 15% of the diet (11,812 mg/kg bw/day for male rats and 11,521 mg/kg bw/day for female rats).

Oxidative Stress Alters the Morphology and Toxicity of Aortic Medial Amyloid.

The aggregation and fibril deposition of amyloid proteins have been implicated in a range of neurodegenerative and vascular diseases, and yet the underlying molecular mechanisms are poorly understood. Here, we use a combination of cell-based assays, biophysical analysis, and atomic force microscopy to investigate the potential involvement of oxidative stress in aortic medial amyloid (AMA) pathogenesis and deposition. We show that medin, the main constituent of AMA, can induce an environment rich in oxidative species, increasing superoxide and reducing bioavailable nitric oxide in human cells. We investigate the role that this oxidative environment may play in altering the aggregation process of medin and identify potential posttranslational modification sites where site-specific modification and interaction can be unambiguously demonstrated. In an oxidizing environment, medin is nitrated at tyrosine and tryptophan residues, with resultant effects on morphology that lead to longer fibrils with increased toxicity. This provides further motivation to investigate the role of oxidative stress in AMA pathogenicity.

Alergia alimentaria / Food allergies

Las reacciones alérgicas incluyen un amplio espectro de reacciones clínicas. Con una incidencia del 15–30% de las enfermedades alérgicas y afecta a un 8% de los niños y específicamente a la proteína de la leche de vaca entre un 5–15% de los niños. Pueden manifestarse como intolerancia o alergia dando manifestaciones respiratorias, cutáneas o gastrointestinales que pueden ser inducidos por cantidades minimas de la ingesta deproteína y cuyo tratamiento básico es la exclusión de la proteína de leche sobre todo en los primeros años de vida.

Allergic reactions include a wide spectrum of clinical reactions. With a 15-30% incidence of llergicdiseases, affecting 8% of children and specifically to the protein in cow's milk 5-15% of children hey can manifest as intolerance or allergy giving respiratory manifestations, gastrointestinal or skin can be induced by minimal amounts of protein intake and whose basic treatment is the exclusión of milk protein specially inthe early years of life.

Structure-function characteristics of the biomaterials based on milk-derived proteins.

There is an impetus on development of implantable biomaterials with the characteristics of improved biodegradability, bio-absorbability and wound healing activities. The milk proteins have valuable nutritional and biological properties, which lead to the promotion of quality health. In this study, whey protein isolate or WPI (highly aggregated) and its component lactalbumin (less aggregated) were melt blended with polycaprolactone (PCL) and then compression moulded into thin sheets ( approximately 1mm thickness). The effects of structural morphologies of the proteins on the mechanical, morphological, in vitro enzymatic degradation, and cytotoxicity and cell proliferation characteristics of the biomaterials were examined. In general, the tensile strength and modulus of the biomaterials decreased with increasing protein content. Compared to WPI, lactalbumin showed a better compatibility with the PCL matrix as observed in the mechanical properties and scanning electron microscopic morphology. The biomaterials exhibited a good retention of the mechanical characteristics after digestion in a physiologically simulated fluid containing trypsin enzyme. However, lactalbumin containing biomaterials showed a better retention of the tensile properties compared to WPI containing biomaterials. The cell culture studies indicated that the biomaterials have no cytotoxic effects, moreover they enhanced the proliferation of L929 cells compared to the pure PCL. Finally, this study indicated that the PCL based biomaterials with a protein content of 20wt% may be applied in fabrication of implantable devices for soft tissue engineering, where it requires a reasonably low to moderate mechanical strength (e.g., approximately 10MPa tensile strength), and improved biodegradability, biocompatibility and tissue healing activities.

In vitro and in vivo safety studies of a proprietary whey extract.

A proprietary whey growth factor extract (WGFE) or Lactermin (Lact milk; ermin growth factors) is a whey fraction of milk containing the major proteins lactoperoxidase and lactoferrin, together with a variety of minor proteins and peptides such as the growth factors IGF-I, IGF-II, PDGF, FGF, TGF-ss and betacellulin. This growth factor component of milk has been suggested to possess biological properties such as the promotion of tissue repair and anti-inflammatory activity. In this study the safety of Lactermin has been evaluated using genotoxicity assays (Ames, mouse lymphoma and micronucleus assay) and in a subchronic (13 week) rat oral toxicity study. In vitro Lactermin did not show any mutagenic properties in the Ames or mouse lymphoma assay and in vivo did not show any adverse clinical effects or in the bone marrow of male or female mice. In the subchronic oral toxicity study in which 10 rats per sex were fed Lactermin mixed with rat diet to deliver doses of 300, 1000 and 3000 mg/kg/day for 13 weeks, male and female rats did not show any test article-related clinical observations or effects on body weight, food consumption, ophthalmic effects, functional observational battery, organ weights, locomotor activity, hematology, serum chemistry, urinalysis or macroscopic or microscopic pathology. The results from the genotoxicity studies and the subchronic oral toxicity study suggest Lactermin is safe for consumption with a no-observed-adverse-effect level (NOAEL) of 3000 mg/kg/day.

Safety evaluation of a milk basic protein fraction.

Milk products are widely consumed by individuals in the US population in the form of fluid milk and milk-derived products and ingredients. Milk is a good source of calcium, which plays a role in maintaining bone health. In addition to calcium, the whey protein fraction of milk contains basic proteins that have been demonstrated to increase bone metabolism and inhibit bone resorption. A specific basic protein fraction in milk (Milk Basic Protein; MBP) was tested in an acute oral toxicity study, teratology study, subchronic oral toxicity study, and reverse mutation assay and no treatment related adverse effects were found. MBP has been evaluated for its use as an ingredient in food and concluded to be safe for its intended use.

T cells of multiple sclerosis patients target a common environmental peptide that causes encephalitis in mice.

Multiple sclerosis (MS) is a chronic autoimmune disease triggered by unknown environmental factors in genetically susceptible hosts. MS risk was linked to high rates of cow milk protein (CMP) consumption, reminiscent of a similar association in autoimmune diabetes. A recent rodent study showed that immune responses to the CMP, butyrophilin, can lead to encephalitis through antigenic mimicry with myelin oligodendrocyte glycoprotein. In this study, we show abnormal T cell immunity to several other CMPs in MS patients comparable to that in diabetics. Limited epitope mapping with the milk protein BSA identified one specific epitope, BSA(193), which was targeted by most MS but not diabetes patients. BSA(193) was encephalitogenic in SJL/J mice subjected to a standard protocol for the induction of experimental autoimmune encephalitis. These data extend the possible, immunological basis for the association of MS risk, CMP, and CNS autoimmunity. To pinpoint the same peptide, BSA(193), in encephalitis-prone humans and rodents may imply a common endogenous ligand, targeted through antigenic mimicry.

An oral sensitization model in Brown Norway rats to screen for potential allergenicity of food proteins.

We developed an oral sensitization protocol for food proteins for the rat. Young Brown Norway (BN) rats were exposed to 1 mg ovalbumin (OVA) by daily gavage dosing for 42 days without the use of an adjuvant. OVA-specific IgE and IgG responses were determined by ELISA. On an oral challenge with OVA some clinical symptoms of food allergy-like effects on the respiratory system, blood pressure, and permeability of the gastrointestinal barrier were studied. In addition, BN rats were orally exposed to a total hen egg white protein (HEW) extract and cow's milk (CM) and the specificities of induced antibody responses were compared with the specificities of antibodies in sera from egg- and milk-allergic patients using immunoblotting. Animals orally exposed to the allergens developed specific IgE and IgG antibodies which recognized the same proteins compared with antibodies from egg- or CM-allergic patients. Among the various clinical symptoms of food allergy, gut permeability was increased after an oral challenge. In addition, some animals demonstrated a temporary decrease in breathing frequency or systolic blood pressure. The results obtained show that the Brown Norway rat is a suitable animal model for inducing specific IgG and IgE responses on daily intragastric dosing of OVA without the use of an adjuvant. Moreover, local immune-mediated effects on oral challenge are observed. The observation that enterally exposed BN rats and food-allergic patients demonstrate antibody responses to a comparable selection of proteins on exposure to different protein mixtures (HEW and CM) further supports the suitability of the BN rat as an animal model for food allergy research and for the study of the allergenicity of (novel) food proteins.

Zinc and intestinal anaphylaxis to cow's milk proteins in malnourished guinea pigs.

Zinc supplementation could favor recovery from diarrhea in malnourished children. As the recent experimental evidence suggests that oxidative stress and intestinal anaphylaxis may contribute to the intestinal dysfunction associated with malnutrition, we postulated that zinc could act through antioxidant or antianaphylactic properties. Control (C), malnourished (M), and malnourished zinc-treated (MZ) guinea pigs were, respectively, fed a normal 30% protein diet, a low 4% protein diet, and a low 4% protein diet plus 1800 ppm of zinc. Milk proteins were included in the diets to trigger intestinal anaphylaxis. Milk sensitization was assessed by passive cutaneous anaphylaxis (PCA) against beta-lactoglobulin and by intestinal anaphylaxis measured in Ussing chambers by the increase in short circuit-current after addition of beta-lactoglobulin (deltaIsc(betaLg)). Oxidative stress was assessed by intestinal lipid peroxidation. The intestinal secretion was assessed by deltaIsc induced by inflammatory mediators. Malnutrition increased the level of anti-betaLg reaginic antibodies [PCA = 1.19 +/- 0.79 and 0.69 +/- 0.67 log(l/titer) in M versus C guinea pigs, p = 0.07] and enhanced intestinal anaphylaxis (deltaIsc(betaLg)) = 16.4 +/- 9.9 and 9.1 +/- 5.8 microA/cm2 in M versus C guinea pigs, p = 0.07), without inducing intestinal lipid peroxidation. Moreover, malnutrition enhanced significantly the intestinal secretory response to histamine and 5-hydroxytryptamine. Administration of pharmacologic doses of zinc during malnutrition inhibited the increase in milk sensitization induced by malnutrition, both at the systemic [PCA = 0.35 +/- 0.55 log(l/titer) in MZ guinea pigs, p = 0.03 versus M] and intestinal (deltaIsc(betaLg)) = 2.8 +/- 2.5 microA/cm2 in MZ guinea pigs; p = 0.001 versus M) level, and prevented the hypersecretion in response to histamine and 5-hydroxytryptamine. These data suggest that zinc has antianaphylactic and antisecretory properties that may contribute to its capacity to prevent intestinal dysfunction during malnutrition.

Anaphylactic intestinal response to milk proteins during malnutrition in guinea pigs.

We investigated whether sensitization to cow's milk occurs during malnutrition and alters intestinal ion and macromolecular transport. Malnourished guinea pigs received a low-protein diet containing either 4% soy or 4% milk proteins, and well-nourished sensitized controls received 26% soy plus 4% milk proteins. To assess milk sensitization, we measured immunoglobulin (Ig) G and passive cutaneous anaphylactic (PCA) responses to beta-lactoglobulin (beta-Lg) and the intestinal anaphylaxis, reflected by the rise in short-circuit current (delta Isc) induced by beta-Lg in tissues mounted in Ussing chambers. To assess intestinal function, we measured ionic conductance and unidirectional fluxes of -14C-mannitol and -3H-horseradish peroxidase (HRP). In malnourished animals fed milk proteins, IgG, PCA, and delta Isc (beta-Lg) increased more than in well-nourished animals. Ionic conductance and mannitol permeability rose in both malnourished groups. Malnourished animals fed milk proteins also displayed enhanced permeability to HRP. These data suggest that increased paracellular permeability is due to malnutrition per se, whereas increased macromolecular transport seems to require both malnutrition and sensitization. They indicate that intestinal anaphylaxis in response to milk proteins is persistent and even enhanced during experimental malnutrition.

[The effect of fermented hydrolyzed whey enriched with lactates on the morphological structure of the internal organs in laboratory animals]. / Vliianie fermentativno-gidrolizovannoi molochnoi syvorotki, obogashchennoi laktatami, na morfologicheskuiu strukturu vnutrennikh organov laboratornykh zhivotnykh.

Results of pathomorphological investigation of rat organs fed during 1 and 3 months the diet containing 8% of protein as milk whey protein partly hydrolysed by enzymes and enriched by lactates (SGOL-1) are presented. The conducted investigation have not revealed of pathological effect of a product SGOL-1 on morphological structures of investigated internal bodies and tissues of the laboratory animals. A increase of body mass of animals and contents of RNAS in cells of practically all bodies and tissues of the animals were also marked.

[Changes of anaphylactic sensitivity to some food proteins and aflatoxin B1 in guinea pigs]. / Izmenenie anafilakticheskoi chuvstvitelnosti morskikh svinok k nekotorym pishchevym belkam pod vliianiem aflatoksina B1.

The study was made of aflatoxin B1 effect on guinea pig alimentary anaphylaxis to chicken ovalbumin (CO) and pasteurized cow milk (PCM), of CO-specific IgG antibody levels, some serum indices, sensitivity of the animals to LD50 histamine. In response to aflatoxin B1 alimentary anaphylaxis both to CO and PCM became more severe, lethality of the anaphylaxis to CO being in logarithmic relation to aflatoxin B1 dose (p < 0.05); specific IgG-antibodies to CO grew in number; serum total protein increased against unchanged levels of albumins; the activity of gamma-glutamyltransferase inhibited; histamine shock gained severity, its lethality being logarithmically related to the aflatoxin B1 dose. The discussion covers mechanisms underlying the animal allergic reactivity responses to aflatoxin B1.

Effect of iron succinyl-protein complexes on gastrointestinal motility in the fasting dog.

Studying the gastrointestinal motor effects of iron compounds may help to elucidate the mechanism originating the gastrointestinal side effects of frequently reported during martial therapy. The aims of the present study were: (1) to examine the gastrointestinal motor effects of ferrous sulfate (reference compound) and (2) to compare its effects with those of two iron succinyl-protein complexes (ITF 1096 and ITF 282, an iron-albumin and iron-casein complex, respectively). In 6 fasting, conscious dogs, fitted with 8 bipolar electrodes and 3 strain-gauge force transducers along the gastrointestinal tract, gastrointestinal motor activity was recorded. Ferrous sulfate and iron succinyl-protein complexes were administered by an orogastric tube at two dose levels: the lower and higher dose levels were approximately equivalent to 10 and 30 mg/kg as Fe, respectively. In control experiments, 154 mM NaCl, ITF 211 (succinylated albumin) and ITF 297 (succinylated casein) were used. Administration of 154 mM NaCl did not affect gastrointestinal motility nor did it disrupt migrating motor complex (MMC) cycling. ITF 1096 and ITF 282, only at the higher dose, lengthened the MMC period and increased intestinal, but not gastric spike activity. The effects of ITF 211 and ITF 297 were similar to those of ITF 1096 and ITF 282. Ferrous sulfate, at the lower dose, lengthened the MMC period; the higher dose disrupted MMC cycling and induced intense, irregular spike bursts in the stomach and in the small bowel, accompanied, in 3 out of 6 dogs, by a prolonged tonic contraction of the upper small bowel. Four out of 6 dogs vomited after the higher dose of ferrous sulfate. No vomiting was observed with any of the other treatments. We conclude that ITF 1096 and ITF 282 have a markedly better gastrointestinal tolerability than ferrous sulfate.

The effects of long-term soy protein and milk protein feeding on the pancreas of Cebus albifrons monkeys.

Twenty-seven 2- to 4-yr-old cebus monkeys (Cebus albifrons) were fed from infancy purified diets containing lactalbumin, soy isolate, casein or soy concentrate as the sole protein source. Hematologic and clinical chemistry values were similar for all groups. Head and tail portions of each pancreas were surgically removed for histopathologic evaluation and determination of protein, RNA and DNA content, and for trypsin and chymotrypsin activity. Hematoxylin and eosin-stained sections from 26 of 27 monkeys showed normal pancreatic tissue with occasional acinar vacuolation in all diet groups. The remaining animal, one of only two fed soy concentrate, had diffuse interstitial fibrosis of the pancreas associated with mild to moderate atrophy of acinar tissue. Biochemical analyses of the pancreatic biopsies indicated no group differences among animals fed lactalbumin, soy isolate or casein. One of two monkeys in the soy concentrate group showed decreased pancreatic protein, RNA and trypsin concentrations; this was probably due to the fibrosis in this animal. No evidence of pancreatic hypertrophy or hyperplasia, as measured by RNA/DNA and protein/DNA ratios, respectively, was seen in any diet group.

Pharmacological and toxicological studies on an iron succinyl-protein complex (ITF282) for oral treatment of iron deficiency anemia.

ITF282, a soluble iron succinyl-protein complex, orally administered to the rat elevates the concentration of iron in the serum to a greater extent than ferritin. The serum iron increase induced by ITF282 is delayed when compared with ferrous sulphate. The ITF282 absorption process, like that of ferritin, proceeds along the physiological pathways without bypassing the transfer system of the intestinal mucosal cells since no further increase of serum metal is observed when giving high doses of ITF282 to the rat pretreated with a saturating dose of ferrous sulphate. Hypochromic and microcytic anemia induced in growing rats by bleeding and feeding a low iron diet is sensitive to both prophylactic and therapeutic oral treatment with ITF282. Iron deficiency anemia and cardiomegaly induced in suckling rats by feeding the pregnant and lactating dams with the low iron diet are reversed by oral treatment of the dams with ITF282. Comparative investigations of the therapeutic efficacy of ITF282 and ferritin made on uncomplicated iron deficiency anemia show that the drugs, p.o. administered during 4 weeks, are equally effective. Preliminary toxicological data in the rat, after single and chronic administrations, show that ITF282 is well tolerated. These findings prove that ITF282 gives an adequate supply of iron from which to make hemoglobin.