Using phytochemicals to enhance health benefits of milk: impact of polyphenols on flavor profile.

The focus of new product development of functional foods is not only on the associated health benefits but also on the sensory properties of the finished product. A trained panel (n=13) assessed 5 milk-based beverages fortified with added apple or grape seed polyphenols and a control using descriptive analysis techniques. The resulting vocabulary consisted of 16 odor, taste, flavor, and mouthfeel attributes, complete with reference standards and scale intensities. Panelists differentiated between samples using all 16 attributes in the vocabulary (P<0.001). Samples fortified with polyphenols, sourced from grape seed and apple extracts, differed significantly. Bitterness and UHT (ultra-high temperature) odor and flavor were the key drivers of product difference. Relatively small (but still significant) differences were identified between those containing polyphenols from the same plant source but different suppliers. We concluded that both grape seed and apple polyphenols suppress the flavor characteristics of the milk, with apple polyphenols having more of an impact than grape seed polyphenols. In particular, the high level of bitterness in apple extracts has large flavor profile implications for product development.

Stability of antioxidants in an apple polyphenol-milk model system.

The stability of antioxidants in an apple polyphenol-milk model system was examined. The model system consisted of skim milk fortified with pH-neutralised apple polyphenols (AP, 0-200mg per 100ml milk), with or without ascorbic acid (100mg per 100ml milk). Physical and chemical changes were evaluated after thermal treatment (120°C, 5min) and oxidative storage (20°C and 38°C, up to 12 weeks). Antioxidant capacity was determined using both oxygen radical absorbance capacity (ORAC) assay and ferric reducing antioxidant power (FRAP) assay. Significant antioxidant capacity was detected in the presence of milk. Antioxidant capacity was retained during thermal treatment but decreased slowly during storage. The concentration of ascorbic acid decreased rapidly, and was close to zero after 2-week storage at 38°C or 10-week storage at 20°C. The brownness of the polyphenol-milk system increased over storage duration of 0-12 weeks; this effect was retarded by the addition of ascorbic acid. This high polyphenol-milk has demonstrated good physical stability.

A novel alpha-amylase gene is transiently upregulated during low temperature exposure in apple fruit.

An alpha-amylase gene product was isolated from apple fruit by reverse-transcriptase PCR using redundant primers, followed by 5' and 3' RACE. The gene is a member of a small gene family. It encodes a putative 46.9 kDa protein that is most similar to an alpha-amylase gene from potato (GenBank accession M79328). In apple fruit this new gene was expressed at low levels, as detected by reverse-transcriptase PCR, in a number of plant tissues and during fruit development. Highest levels of mRNA for this transcript were observed 3 to 9 days after placing apple fruit at 0.5 degrees C. Phylogenetic analysis of amino acid sequence places the potato and apple proteins as a distinct and separate new subgroup within the plant alpha-amylases, which appears to have diverged prior to the split between monocotyledonous and dicotyledonous plants. These two divergent alpha-amylases lack the standard signal peptide structures found in all other plant alpha-amylases, and have sequence differences within the B-domain and C-domain. However, comparisons with structures of known starch hydrolases suggest that these differences are unlikely to affect the enzymatic alpha-1,4-amylase function of the protein. This is the first report of upregulation of a dicotyledonous alpha-amylase in response to low temperature, and confirms the presence of a new family of alpha-amylases in plants.

Apple beta-galactosidase. Activity against cell wall polysaccharides and characterization of a related cDNA clone.

A beta-galactosidase was purified from cortical tissue of ripe apples (Malus domestica Borkh. cv Granny Smith) using a procedure involving affinity chromatography on lactosyl-Sepharose. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that two polypeptides of 44 and 32 kD were present in the fraction that showed activity against the synthetic substrate p-nitrophenol-beta-D-galactopyranoside. The enzyme preparation was incubated with polysaccharide extracts from apple cell walls containing beta-(1-->4)-linked galactans, and products of digestion were analyzed by gas chromatography. Small amounts of monomeric galactose were released during incubation, showing that the enzyme was active against native substrates. Amino acid sequence information was obtained from the purified protein, and this showed high homology with the anticipated polypeptide coded by the ethylene-regulated SR12 gene in carnation (K.G. Raghothama, K.A. Lawton, P.B. Goldborough, W.R. Woodson [1991] Plant Mol Biol 17: 61-71) and a harvest-related pTIP31 cDNA from asparagus (G. King, personal communication). Using the asparagus cDNA clone as a probe, an apple homolog (pABG1) was isolated. This clone contains a 2637-bp insert, including an open reading frame that codes for a polypeptide of 731 amino acids. Cleavage of an N-terminal signal sequence would leave a predicted polypeptide of 78.5 kD. Genomic DNA analysis and the isolation of other homologous apple clones suggest that pABG1 represents one member of an apple beta-galactosidase gene family. Northern analysis during fruit development and ripening showed accumulation of pABG1-homologous RNA during fruit ripening. Enzyme activity as measured in crude extracts increased during fruit development to a level that was maintained during ripening.

Investigation of usefulness of electroreticulograms obtained from the sheep body surface in estimation of reticulum electrical activity.

The experiments were carried out on 3 sheep with one pair of platinum electrodes chronically implanted in the reticulum wall. The reticulum action potentials were recorded simultaneously from the body surface (using the needle electrodes placed subcutaneously) and from the implanted electrodes using an electroencephalograph Reega XVI Duplex TR. It was found that the two- or three-phase deflections on the electroreticulograms obtained from the body surface were exactly correlated with the series of potential spikes on the records from the implanted electrodes. The number of phases in each deflection was equal to the number of the series of potential spikes during the reticulum excitation. During the reticulum hypotony the decrease in action potentials was observed. That is why we were not able to record the electrical activity from the body surface, while it was still possible to record electroreticulograms from the implanted electrodes.

[Effect of dopamine on cecum motility in sheep]. / Wplyw dopaminy na motoryke jelita slepego u owiec.

The influence of dopamine on the motility of the caecum were studied. The experiments were carried out on 4 sheep with chronic fistulas of the caecum. The contractions of the caecum were recorded by the balloon method. Dopamine infused intravenously at a rate 29-38 micrograms.kg-1.min-1 inhibited the motility of the caecum. Dopamine infused after beta-adrenergic blocker propranolol (0.5-1.0 mg.kg-1) inhibited the movements of the caecum, however administrated after domperidone (0.2-0.4 mg.kg-1) didn't change its movements. It has been concluded that dopamine inhibits the caecum motility in sheep through specific dopaminergic receptors.

[Effect of dopamine on small-intestinal motility in sheep]. / Wplyw dopaminy na motoryke jelita cienkiego u owiec.

The receptors involved in dopamine effect on the motility of the ileum were studied. The experiments were carried out on 3 sheep with chronic fistulas of the ileum and rumen. The contractions of the fistulated parts of the alimentary tract were recorded by the balloon method. Dopamine infused intravenously at a rate 28-38 micrograms.kg-1.min-1 inhibited the motility of the ileum. Domperidone (0.4-0.8 mg.kg-1) did not abolish this inhibiting influence of dopamine. Dopamine infused after propranolol (0.5-0.7 mg.kg-1) inhibited the motility of the ileum, however administrated after phentolamine (0.6-0.8 mg.kg-1) did not change its movements. It has been concluded that dopamine inhibits the ileum motility in sheep through alpha-adrenergic receptors.

Participation of the beta-adrenergic receptor in the enteroruminal reflex and enteroenteric reflex.

In sheep with chronic fistulae of the small intestine and rumen the participation of the beta-adrenergic receptor was investigated in the enteroruminal reflex and enteroenteric reflex using the method of pharmacological analysis. The movements of the segments of the digestive tract with fistulae were recorded by the balloon method. A solution of hydrochloric acid administered into the ileum caused a reflex stimulation of its motor activity and inhibited the movements of the rumen. Intravenous administration of propranolol before instillation of the acid into the intestine abolished or reduced greatly the reflex inhibition of the movements of the rumen and in the small intestine it enhanced significantly the studied reflex reaction. Thus stimulation of the beta-adrenergic receptor plays an important role in the reflex stimulation of the motor activity of the rumen, and stimulation of the motor activity of the small intestine in the enteroenteric reflex is limited by the effects derived from this receptor.

The phenomenon of reversal of reticulum action potentials recorded from the body surface of sheep.

The experiments were carried out on sheep with reticulum fistula. The biopotentials and transformed mechanogram of the reticulum were recorded simultaneously on the body surface with an electrocardiograph. Lactic acid solution 1.6 mol/l was administered through the fistula into the reticulum in an amount of 15 mmol/kg of body weight. It was found that administration of this acid caused reversal of the biopotentials of the reticulum. This phenomenon lasted from 10 to several scores of hours and occurred with pH of reticulum contents below the normal range as well as after return of normal pH.

Interaction between bacterial metabolites and some pesticides. II. Change of phytotoxicity of the herbicide Roneet by the phenolic metabolites of Arthrobacter sp.

The bacteria from Arthrobacter genus isolated from sugar beet rhizosphere were found to produce gallic, protocatechuic, p-hydroxybenzoic, syryngic, vanillic, veratric acids, p-quinone and two unidentified phenolic compounds. The mixture of the bacterial phenolic compounds increased the phytotoxicity of Roneet, inhibiting the germination of wheat. Model experiments showed that the phenolic acids used with the herbicide Roneet increased its phytotoxicity and p-hydroxybenzoic acid was found to be the most active. It was found that in the presence of the phenolic acids, particularly of p-hydroxybenzoic acid, the herbicide content in the seeds of wheat was reduced. The UV and IR spectra of equimolar amounts of p-hydroxybenzoic acid and Roneet showed the hydrogen bond between H of carboxylic group of acid and O of C=O group of the Roneet molecule. It could be concluded that Roneet conjugated with p-hydroxybenzoic acid was more phytotoxic than the initial compounds.

Analysis of the role of adrenergic receptors in the regulation of small intestine motor activity in sheep.

In sheep with chronic fistulae of the small intestine and rumen the participation of alpha- and beta-adrenergic receptors in the regulation of the motor activity of the small intestine was studied by the method of pharmacological analysis. The movements of the fistulated parts of the alimentary tract were recorded by the balloon method. Slow intravenous infusion of isoprenaline inhibited the contractions of the small intestine. This inhibitory effect of isoprenaline was abolished by propranolol. Intravenous phenylephrine inhibited the motor activity of this intestinal part as well. The effect of phenylephrine was abolished by pretreatment with dihydroergotamine. In the small intestine of sheep stimulation of the alpha and beta adrenergic receptors decrease the motor activity of intestine.

Participation of the adrenergic receptors in the regulation of large bowel motor activity in sheep.

In sheep with long-standing fistulae of the caecum, proximal loop of the colon and rumen the motor activity of these parts of the intestine was studied using the balloon method. Slow isoprenaline infusion caused first inhibition of caecal contractions, followed by gradually decreasing intensity of contractions of the colon and rumen. This inhibitory effect of isoprenaline was eliminated by propranolol. Intravenous infusion of phenylephrine failed to inhibit the colonic motor activity. A small dose of atropine, similarly as isoprenaline, caused an irregular inhibition of the contractions of the caecum, colon and rumen. In sheep, in contrast to other animal species, the effects inhibiting large intestine motility are mediated only by the beta-adrenergic receptors. The inhibitory effect of the beta-adrenergic receptors was strongest in the caecum.

Nervous regulation of abomasum motor activity in sheep.

Pharmacological studies of abomasum motor activity were carried out in 3 sheep with chronic fistulae in the rumen and the fundus and pyloric part of the abomasum. The contractions of these parts of the stomach were recorded by the balloon method. The obtained results showed that in the abomasum stimulation of alpha-adrenergic receptors raises the degree of motor activity while stimulation of beta-adrenergic receptors inhibits it. It has been shown also that the sympathetic system has a greater influence through the beta-adrenergic receptors on the motor activity of the pyloric part than on that of the fundus of the abomasum. The analysis of the records obtained after bilateral vagotomy demonstrated that the automatism of the pyloric part was greater than that of the fundus of the abomasum.

Effect of microorganisms on the phytotoxicity of herbicides. VI. Modification of venzar activity by bacterial cultures.

Interactions between two bacterial strains and venzar were compared. It was found that the mechanism of interactions is various and causes the modification of herbicide phytotoxicity. Metabolites of Bacillus subtilis 72 interfered with herbicide by affecting physiological processes in plant tissues and enhancing its inhibitory influence. Arthrobacter sp. 18 strain decreased the phytoinhibitory effect of herbicide due to conjugation with the carrier from venzar.

Motor activity of the abomasum in sheep.

The experiments were carried out on 5 sheep with chronic fistulae of the rumen and of the fundus and pyloric part of the abomasum. The motor activity of the parts of the stomach containing fistulae was recorded by the balloon method. The analysis of the obtained contraction curves showed that the motor activity of the pyloric part was more intense than that of the fundus of the abomasum, and the intensity increased with filling of the multichamber stomach with food, food intake increased the tonus and the intensity of peristaltic movements in the fundus and pyloric part of the abomasum, rumination had no effect on the motor activity of the fundus but increased usually the frequency of contractions of the pyloric part. It has been demonstrated also that the movements of the pyloric part of the abomasum can be inhibited for short time periods.

Regulation of motor activity of omasum after blockade of vagus nerves.

In 3 sheep with subcutaneously exposed vagus nerves and with chronic large fistula of the rumen pharmacological analysis of the motor activity of omasum was carried out after blockade of vagus nerves. The contractions of omasal canal, reticulum and rumen were recorded by the baloon method. After blockade of the vagus nerves with anesthetics the proper pharmacological agents were administered in intravenous injections. In the light of the obtained results it was found that contractions of the omsal canal, persisting after vagal blockade, are independent of the influence of the autonomic system and they are probably due to myogenic automatism. It has been also demonstrated that both alpha and beta adrenergic receptors participate in regulation of the tonous of omasum musclular layers.

Microorganisms as indices of environmental pollution by smelting industry.

The aim of the study was to prove suitability of some microbiological tests for determination of the degree of soil pollution by copper industry. The microbiological tests reveal sensitivity to metal compounds present in dusts emitted by smelters. The sensitivity of the selected strains to the dust present in the agar medium and soil has been determined and 30 strains, of which 50% reacted to the presence of 1--2 g of the dust in medium, were recommended for the tests. The tests showed accumulation of heavy metals contained in the dust mainly in the upper soil horizons and the highest pollution with the dust approximately 800 m away from the emitor in northeastern direction, which is the direction of prevailing winds of that area. The results obtained are in accordance with theoretical assumption as well as with the literature based on chemical analyses of polluted soil.

The effect of microorganisms on phytotoxicity of herbicides. III. Interaction of Bacillus sp. 72 with Venzar.

Interactions between Venzar and some metabolites of Bacillus ps. 72 were studied. This strain was found to produce flavonoids increasing the phytotoxicity of Venzar. Venzar action was also increased by NH4+ ions released aboundantly by the bacterial strain.