Biochemical Transformations of Lipide and Carbohydrat-Protein Nano Complex in Liquid Foodstuff

The process of enzymatic hydrolysis of dairy whey at presence of a polyfermental preparation pancreatin was investigated. The process of hydrolytic transformations of dairy proteins, fats and carbohydrates in comparison of a parallel estimation of changes of the sizes of the lipid, carbohydrates-protein formations which are being in nano region of area was studied.


Introduction
Fibers, fats and carbohydrates are the major components of foodstuff. They form internal structure, both liquid, and firm food compositions, and, in case of liquid products what milk and dairy whey, formation of internal structures is, for example, is reduced basically to emulsify to the condition of substance formed under laws of colloid systems [1]. Dairy whey -a valuable food stuffs, especially for older persons, because of presence in it the serum fibers containing in the structure greater, than in casein, quantities of irreplaceable amino acids. These fibers are high-grade and are used by an organism for a structural exchange, therefore dairy whey draws the increasing attention as raw material for reception of functional food stuffs [2,3].
By manufacture of dairy whey in it half of dry substances of milk, including the most part of lactose and mineral substances passes on the average. The basic component of dry substances of dairy whey is lactose which mass fraction makes more than 70 % of dry substances [4]. During fermentation and the subsequent biochemical transformations of components, the structure of an albuminous and carbohydrate part of a product undergoes a number of the transformations affecting nutritional value. Presence of components of milk and the common biological properties of whey allow to carry it to valuable industrial raw material which can be processed in various useful components [5].
Traditional dairy products represent steady or unstablecolloid-lipidic systems with protein-carbohydrate environments. The sizes of the basic formations in liquid structure of fresh dairy products, make, as a rule, less than 100 nanometers, that formally allows to carry such objects to systems with nano particles [6].
As definition of dispersiveness of liquid nano systems is enough a complex experimental problem, was of interest to estimate the possible changes of nano clusters in a liquid product-whey of milk during known biochemical transformations of the basic most valuable components.
The purpose of work consist in a quantitative estimation of the sizes carbohydrates-protein nano clusters during biotransformation of dairy raw material at a pseudo-molecular level.
We used the pharmacopeia pancreatin with the common proteolytic activity of 12500 U/mg, lipolytic activity of 1000 U/mg and amilolytic activity of 12000 U/mg [7].
For the control of the sizes carbohydrates-protein nano clusters used the Rayleigh scattering data allowing on spectral characteristics of disperse system to estimate turbodimetric the linear sizes of particles in liquid system [8].
For suspension with spherical particles it is possible to write The Rayleigh equation in the form of I Σ / I o = 24π 3 /λ 4 ·[( n 1 2 -n 2 2 )/(n 1 2 + n 2 2 )] 2 ·С V ·V, where: I Σ -full intensity of light disseminated of 1 sm 3 of system in 1 sec; λ-length of a wave of light, sm; n 1 -a parameter of refraction of a disperse phase (it is equal 1,333); n 2 -a parameter of refraction of a dispersive phase (accepted equal as for lipide dairy fat n D 20 = 1,5); С V -a volume fraction of a disperse phase; V -the volume of a particle, sm 3 . Considering, that the turbidity τ = I Σ / I o is numerically equal to the light of energy disseminated of 1 sm 3 of a solution in all directions, and also that the Rayleigh equation is carried out for very diluted systems, [τ] = lim (τ/ С V ), С V → 0 in work used the diluted water dispersions of 1:2000 … 1:10000 at = 546 nanometers for construction of dependences of ratio of τ / С V from С V . In the given coordinates extrapolation at С V → 0 established τ / С V and further calculated approximate diameter of investigated particles how it is described in works [9,10]. The structure of carbohydrates (CH) was studied with use the BioLC chromatographic system including gradient pump GS50, electrochemical detector ED50, the the generator of eluent EG50 about 10 mm NaOH, chromatographic oven LC25 with column CarboPac PA20, manufactures DIONEX (Germany) [11,12]. Definition of the contents of free carbohydrates carried out in water extracts of 0,01 g of the sample (or 100 mkl of liquids) in 100 ml of HPLC water / 0,45mkm filter at 25℃. As standards of carbohydrates we used: arabinose (Ara, D- , water solutions with concentration of 0,001 mg/ml. The analysis of structure lipids was spent on modified Folch method [11,13] for this purpose the 1 − 5 ml of the whey, containing of 10 − 20 mg of lipids evaporated by means of using of the rotor evaporator dry and subjected the methylation. For comparison with lipid of milk the 20,0±0,1 ml of the cow milk mixed about 50 ml of a mix (1:1) of chloroform with methanol and further aliquot from a chloroformic layer subjected evaporation as is specified above.
For maintenance of completeness of definition of all fatty acids (FA), containing in lipid fractions the gas chromatography analysis, samples of the allocated fats subjected methylation on the modified method [11,14]. For this purpose 0,01 g of lipids in a mix of 15 % of a solution of 3 ml acetyl chloride in a metazero boiled at 100℃ during 2 h with the subsequent neutralization of a reactionary mix by addition of 1,25 ml of saturated solution of КОН of in СН 3 ОН up to рН 5.0 -6.0. In a mix added 3 ml of saturated water solution of NaCl and 3 ml of hexane, mixed, defended during 30 mines and selected on the analysis of a solution of 0.5 ml from transparent hexane a layer containing methyl ethers of FA. Ethers analyzed on gas chromatograph HP6890 Hewlett-Packard (USA) with PID detector and capillary column HP-Innowax 30mmx0,32mmx0,5mkm in a current of nitrogen. The programmed rise in temperature with a speed 10℃ . min -1 columns with 100 up to 260℃, injector and the detector -up to 250 and 300℃, input 1 mkl of tests, a stream of hydrogen from the generator -35 ml . min -1 , nitrogen -20 ml . min -1 , split mixture 1:100 was provided. Identification of peaks spent with use of the standard of FA (methy l) : cis-13, 16 Quantitative calculation spent with use of the automatic program of processing of chromatographic data for С 6 -С 24 FA Winpeak (Germany). Identified in three repetition peaks FA which maintenance of exceeded of 0,01% from a total sum.
Free FA analyzed a usual method by distillation of about the ferry, reextraction of components into hexan with the subsequent analysis of structure to method of a gas chromatography similarly specified above, but from chromatoweights-spectrometer detector MSD 5975 under control of Agilent MSD ChemStation and performance of library search for the quantitative analysis on database NIST08.L of Agilent (USA).
Degree of hydrolysis of fiber estimated on change of nitrogen of free amino groups [16]. Analyzed of lyophilic dried up hydrolyzates in comparison with milk and dairy whey. As is known, for improvement of biological properties of dairy whey apply the enzyme-galactosidase to transformation of lactose into more sweet both well soluble and acquired mix of monosugars (glucose and galactose), and also for hydrolysis of fibers up to peptide and amino acids (increase in biological value and quality -a transparency and absence of a deposit) proteolytic enzymes: protease Actinomyces vulgaris, tripsin, protophradin, pancreatine, chicken pepsin, protoalbin, that provides 50−90% conversion of dairy fiber [17,18].

Results and Discussion
Earlier in our works, etc. researchers it has been shown, that pancreatine a pancreas of pigs and large horned livestock is the polyfermental preparation possessing the protease, lipase and amylase activity and actively participates in biotransformations of CH, fats and fibers in alive organisms [19][20][21].
On fig. 1 the curve of dependences of a degree of hydrolysis of dairy whey on parameters of process are presented. It is visible, that in case of used pharm. pancreatine the optimum degree of hydrolysis of albuminous components makes: рН 6.8, temperature 35-40℃, time 6 h at concentration of enzyme of 2% to weight of whey. In these conditions the degree of conversion of the basic components exceeds 80%.
In tabl. 1 data on hydrolyses are presented to disintegration of fibers of dairy whey. It is visible, that enzyme hydrolyzate of milk with a high degree of hydrolysis contains not only a greater share of free amino acids, but also parallel decrease in the sizes of dairy particles which as a result of hydrolysis decrease practically more, than twice is observed. The size of the size of particles which in dairy systems represent lipide carbohydrates-protein macrocomplexes, is important enough factor, especially at reception of functional foodstuff with the raised bioavailability in a treatment-and-prophylactic meal. With a high degree of dispersive ability of components, especially at a level nano, apparently, it is possible to use products for creation of substitutes traditional parenteral compoundings.
The research problem was attempt to track the transformation of nano claster, also interesting studying of change of a chemical compound of the basic components however was represented. As is known, in dairy products some hundreds FA which are connected in triglyceride, forming dairy fat contain [22][23][24].
However the basic is fat-acid structure includes no more than three tens FA, major of which (more than 80%) are palmitic, stearic and oleic (tabl. 2). The influence of polyfermental preparations with lipolytic activity leads to that the part of triglyceride breaks up with liberation corresponding FA. The traditional researches connected with hydrolysis of milk and dairy products are usually directed on transformation of dairy fibers and CH.
From tabl. 2 it is visible, that the mass fraction of low fat acids С6 -С12 during biotransformation decreases, and the contents of higher limiting fat acids С14 -С22 increases. Presence of С4 -C8 FA in total amount more than 5 -8% is prominent feature of dairy products and the cow milk and these FA, apparently are most subject to influence of enzymes. Transformations of nonsaturated fatty acids carry more complex, as a rule, extreme character. Similar character in change of is fat-acid structure of animals lipids a various origin, we observed of investigating process of storage of production on the basis of meat raw material [25,26].
In tabl. 3 the results of definition of amount of minor CH in processable products are presented.
Tabl. 3 shows the analysis of data, that a carbohydrate component dairy nano clasters, apparently, is the most unstable group of substances. Their contents in an analyzed mix during enzyme processings decreased practically in thirty times. Thus the mass fraction of the core dairy disaccharidelactoses decreased from 91-97 % more, than three times that can specify sufficient activity of pancreatin during hydrolysis of dairy sugars.

Summary
Thus, the enzyme processings of dairy components leads to degradation of all making substances of milk and change of their componental structure with simultaneous reduction of the sizes of lipid, carbohydrate and protein formations, which size are in nano scale areas.