Gladky F. F., DPhil, professor, Chumak O.P., candidate of technics, DPhil, professor, Markov K. V., junior research fellow, Gasuk K. V., senior staff scientist,. Usichenko O. V, master

National Technical University “ Kharkov polytechnic institute”, (Ukraine)

To question about enzymatical technology of modification and refining of fats

Transformation of accompanying to fats substances (phospholipids, fatty acids) using enzymatic preparations was studied and the results of such transformations were analysed. During the research there were determined new ways for developing of refining methods, main regularities in fats (acylglycerines) enzymatic transformation like ethanolysis and hydrolysis. Modifying of acylglycerines was carried out by their interesterification with the appropriative esters using specific lipolythic enzymatic preparations, that allowed producing of special confectionary fats of high quality.

Рассмотрены результаты исследований преобразования сопутствующих жирам веществ (фосфолипидов, жирных кислот) с помощью ферментных препаратов. Определены новые пути развития методов очистки жиров, основные закономерности преобразования жиров (ацилглицеринов) под влиянием ферментов, в частности етанолиза и гидролиза. Осуществлено модифицирование ацилглицеринов путем переэтерификации с этиловыми эфирами соответствующих жирных кислот при участии специфических липолитических ферментных препаратов, что дало возможность получения специальных кондитерских жиров высокого качества.

Presently in Ukraine as wells in other countries, fats are refined from accompanying substances with traditional methods which are based on usage of alkalis, acids, etc.

A part of accompanying substances are extracted from fats in vacuum/ This process is highly energy consuming and produces waste substances. Thus, studing and research of substances accompanying to fats is an important problem.

The substances which accompany to acilglycerines in fats are: fatty acids, waxes, phospholipids, coloration, taste- and odour related substances: sterols, non-specific accompanying substances.

The object to research is a group of activated and water soluble enzymes, both specific and non-specific, and substances accompanying to fats like phospholipids and fatty acids.

All the exiting methods and technologies of enzymatic hydrotation are based on the reaction of oil phospholipid hydrolysis usingphospholipases hydrolase subclass enzymes (according to the international classification К. Ф 3.1).

They catalyze the the process of transforming of phosphorous-containing compounds (contained in fats, so-called phosholypids), i. e. stimulate the reactionof hydrolysis of ester-link in a phosholypid molecul. Among them are, phospholipases A₁, A₂, C and D which differ from each other in their ways of influencing the substrate.

The fundamental mechanism of enzymes influencing phospholypids looks like:

where R₁, R₂ – saturated or unsaturated oil carbonic remainder of fatty acids;

X – hydrogen, nitrogen base or polyol remainder.

Phospholipases A₁ (К.Ф. 3.1.1.32, phosphatid-1-acilgidrolases), catalyze hydrolysis of phospholypid molecule’s ester-link in α-position. They are contained in pancreatic gland juice of animals and people [1].

Phospholipases A₂ (К.Ф. 3.1.1.4, phosphatid-2-acilgidrolases), which catalyze hydrolysis of phospholypid molecule’s ester-link in β-position are divided into 3 classes: venom enzymes (insects, snakes, scorpions, bees, etc); grass enzymes; intracellular which are contained in plants and microorganisms tissues [2]. They differ substantially in structure, substrat specificity, biological effects, and physicochemical properties [3]. An active and the most studied enzyme of this type is contained in venoms [1-4].

Phospholipases A₂ (К.Ф. 3.1.1.4.3, catalyze hydrolysis of molecule’s ester-link between diacylglycerin and substitute phosphoric acid; well-known phosphatidilholine-holinephosphohydrolasa and phosphatidilinizitol-inozitolhosphohydrolasa) are contained in bacterial toxins. It was found in tissues of animals, including brain tissues in little quantities in bound state [1,5,6].

Phospholipases A₂ (К.Ф. 3.1.4.4, catalyze hydrolysis of molecule’s ester-link between phosphatic group and alcohol in a phospholipid molecule) is contained in plants’ tissues (cabbage, roots of radish, cotton plant seed, etc), as well as in tissues of some animals like rats.

After having analyzed an assortment of phoshpolipase and having studied a proposition of enzymatic specimens on the market , it was decided to choose the industrial phospholipase A specimens (produced by Denich “Novozymes” company) and available in Ukraine titled phospholipase.

The target was to: 1) define the conditions for using hydrolyze-type enzymes for producing phosphoypidic lysoforms and etherification of fatty acids, contained in fat; 2) identifying main regularities of fat accompanying substances bioconversion.

The main scientific results of the performed research consist in identify regulation of substances accompanying to fats – phospholipids and fatty acids using lipolitic enzymes. Also there were calculated content and composition of phospholypids and fatty acids in new breeds of sunflowers, provided by Ukrainian Academy for agricultural sciences.

It was investigated kinetics of phospholipids hydrolysis and the maximal speed of this reaction was calculated as well as effective temperature and Michaelis constant.

It was proved scientifically that mainly lysophosphoydilamins and lysophosphoydilcholin content is increased after enzymatic hydrotation.

Also it was proved a possibility of fatty acids(which are contained in sunflower oil) by ithanol. This fact makes possible creation of a new foodstuff, i. e. oils enriched by easy digested components (ethyl esters of fatty acids).

Authenticity of scientific results was confirmed by data obtained in industrial conditions at Pology oil-extracting plant.

It is known a method of refining oils from fatty acids by distillation under reduced pressure and high temperatures. The imperfections of the method are appearing of dark-colored substances having unpleasant smell, and highly energy-consuming.

There is a method of oils and fats refining when they are treated with alkaline agents, for example, by sodium hydroxide water solution under the temperature of 40 - 90°C [7].

Imperfection of the method is that fatty acids (which are refined, when being processed by alkaline agent) are transformed into fatty acids salt – soaps. During this process, water solutions of soaps are formed which emulsify oil or fat, creating fat – containing yield of production process – a soapstock. A mass of soapstock increases when oil’s acidic number is increased. In addition, sodium hydroxide is a dangerous substance that requires specific measures for safe using of the method.

The goal was to simplify the technology of oils and fats rectification using enzymes. And the goal was achieved by using glycerin (instead of alkalines) which was used for treating oils and fats in presence of specific lipolytic immobilized (or non-immobilized) enzyme, kipping the mass proportion of oil or fats: glycerin: enzyme equal to 100: 0,5÷5 : 0,1÷10, the temperature is 30 – 80°C, pressure is 20 – 50 mm Hg vacuum during 0,5 – 15 hours.

After enzymatic refining, acidic number changes for oils is shown in table 1.

Table 1 – Changes of acidic number for oil using enzymes

№	Enzyme	Initial acidic number, mg KOH/g	Final acidic number, mg KOH/g	Time of reaction, hours
1	Lypozyme RMIM	4,5	0,3	7
2	Novozeme 435	5,2	0,2	3,5
3	Novozeme 435	5,8	0,25	7
4	Lypozyme TLIM	3,0	0,3	6

Advantages of such a refining method are; single-stage refining; fatty acids are not extracted from oils or fats buy are transformed into acylglycerins; lack of wastes; degreased losses of product oil or fat; glycerin as an agent, is much more ecological and safe stuff comparing to o-phosphoric acid and caustic soda.

A patent №30031 “Method of oils and fats refining” was issued on 11 Feb 2008.

A particle value of performed research is outlining of new ways of development methods refining fats from accompanying substances using enzymes.

It has to be said, that now in Ukrain, as in other CIS countries producing of foot supplement – ethyl esters of fatty acids, mono- and diacylglicerins, which are classified as substances for improving food stuff quality, is performed using chemical agents (alkaline, acids etc).

The technology of such products is quite complicated and energy consuming. More than that, refining of final products leads to appearing of essential amount of waste products. For example, mono- and diacylglicerins synthesis requires using of special equipment (target monoacylglicerins fraction is extracted by molecular distillation) [8,9].

That is why researching of fats transformations by using enzymes, and finding conditions for producing target products using lea energy consuming methods and high yield are actual and necessary.

The researches’ goal was to determine fundamental laws of fats (acylglicerins) transformation under influence of enzymes, particularly ethanolysis and hydrolysis.

The final goal of design was implemented by the following steps:

· appraisal of properties of enzymatic preparation produced industrially or having experimental-industrial status;

· determining of external factors influencing the parameters of the appropriative reactions;

· determining of optimal conditions for obtaining esters of fatty acids and incomplete acylglycerines.

Stated below are the results of this scientific research.

It was proved a possibility of using enzymatic preparation on the carrier Lipozym TLIM, Lipozym RMIM and Novozym 435 to transformation of acylglycerines (fats), particularly alcoholysis, aided by mono- and polyatanomic alcohols.

It was revealed that during ethanolysis reaction, the highest level of triacylglicerins transformation using Lipozym TLIM is 82% (weight), using Lipozym RMIM is absolute 80% (weight) and the highest level of transformation is 94% (weight) when using Novozym 435.

It was found that during glycerolysis reaction the transformation level os triacylglycerins using Lipozym TLIM is 34,4% (weight), using Lipozym RMIM – is 17,4% (weight), and the highest level of transformation is 85,5% (weight) when using Novozym435.

The optimal conditions for alcoholysis are determined as: period length for the most high speed reaction – 2 hours; tge temperature for Lypozym TLIM and Lypozym RMIM is 30 – 35°C, and for Novozym – is 60 - 70°C;components proportion is stoichiometric.

Posibility of the pbtained results is proved by their correlation on conditions using different measuring ways. For example, the data related to the reaction product composition, calculated using thin-film chromatography, are proved by dividing those product on adsorbing columns, by measuring viscosity, refraction factor, and using chemical methods.

Also important is the problem of detection of main regularities of enzymatic interesterification implemented to ethers of mono- and polyatomic alcohols as a method of fats modification.

Promising and timeliness of research work is proved by the possibility of creating new fats – related technologist based on the obtained results. Such technologies are also important for confectionary industry.

For the moment , such fats are imported from abroad.

Scientific actuality of research is proved by absence of important related to interesterification of esters of mono- and polyatomic alcohols in special literature.

Producing of high quality confectionery fats possible by multifunctional fractionating of palm oil and other oils. This method is labor-intensive and requires sufficient power inputs. Using partky-hydrogenizated oils (for example, sunflower oil, rape oil, etc) as special confectionary fats does not allow to produce high- quality confectionary. In addition, there is a considerable quality of trans-isomers in such fats. Such substances, in the judgment of some specialists, shoud be of limited quantities in foodstuff.

One of the feasible ways of resolving the problem is acylglycerins (fats) modification using specific lypolotic enzymes.

We carried out acylglycerins (fats) modification by their interesterification with ethyl ethers of the appropriate fatty acids using specific fat-splitting enzymatic preparations. This method allows to obtain symmetric acylglycerine, and also asymmetrical acylglicerines when using mixtures of esters.

A patent №79728 «Method of obtaining modified fats” was registered for this technology. The research was awarded whit a gold medal at the III International show of inventions and new technologies “New time” in Sevastopol city.

Analysis if the research proved the advantage of enzymatic technologies: production cost price redaction, ecological compatibility of manufacturing, possibility of obtaining products for various purposes, creating of waste-free technologies, and etc.

The particular value of the research consists in rational design and deoploing into industrial production new energy- - and recourse-saving technologies for producing: esters as food additives, and as a fuel component for internal-combustion engines; monoglycerines which are used as food surface-active agent fats; fats enriched with diacylglycerines as a component of foodstuff – salad oils, margarines, spreds, confactionary fats for special purposes, etc.

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3. Литвиненко Н.М., Кисель М.А. Эндогенные фосфолипазы А₂: Структура и функция. – Минск: Навука і техніка, 1991. – 270с.

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