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ПРОМЫШЛЕННАЯ МИКРОБИОЛОГИЯ И ЭКОЛОГИЯ
UDK 664
E. V. Nikitina, L. Z. Gabdukaeva, M. S. Egkova
THE EFFECT OF ENZYME MODIFIED STARCHES ON THE QUALITY AND BIOSAFETY OF MEAT PRODUCTS
Key words: Enzyme modified starches, quality of meat stuffing and products, bio-safety products.
Potato starches obtained after the enzyme treatment was used to obtain minced meat products. Functional and technological and physico-chemical parameters of products with fermented starches are not inferior model samples with chemically modified starches. The use of fermented starches in the production of meat minced products have a preference at biosafety and environmental parameters, as revealed lower levels of genotoxicity of water-soluble and aceton-soluble fractions..
Ключевые слова: мясные и рыбные полуфабрикаты, полисахариды, функциональное питание.
Картофельные крахмалы, полученные после обработки ферментами, использовались для получения продуктов из рубленого мясарубленого мяса. Функциональные и технологические и физико-химические параметры продуктов с такими крахмалами не уступали образцам с химически модифицированным крахмалами. Использования ферментно модифицированных крахмалов в производстве рубленых мясных продуктов предпочтительнее с точки зрения биологической безопасности, так как у них был выявлен более низкий уровни генотоксичности водо- и ацетон-растворимых компонентов готовых изделий.
Introduction
Modern food manufacturers are actively using various kinds of starches in their technologies, including modified starches. The potato starch is used most widely in the meat processing industry because it have the low gelatinization temperature (68-72 OC).
The degree of change in the granular structure of starch is strongly dependent on potato varieties of which the starch is selected. It has been found that potato starch granule size are located in a large range from a few microns in diameter - for small pellets to 110 ^m -for large pellets [1]. Potato starch is a good structurant, it has a good swelling properties, the ability to stabilize of dispersions (bind water and fat), showing good adhesion and rheological properties. However, food industrial use of native starches is limited of their tendency to retrogradation and syneresis exposure [2].
Besides their native form like gels, pastes from cereal starches, waxy maize starch, in which up to 99% of amylopectin [3] tends to degradation on prolonged heating, vigorous stirring or by the action of acidic conditions.
In recent years modified starches are applied more and more in the food industry. Properties of those starches differ significantly from native starches as a result of a variety of processing methods (physical, chemical, biological). Modified starches have better moisture-retaining properties [4, 5], viscosity and structuring properties [6, 7, 8, 9].
They can also affect the number of critical parameters of the end product such as yield, taste, texture, shelf life, and others. It should be noted that the Joint FAO/WHO Expert Committee on Food Additives recommends as far as possible to exclude the use of chemically modified starches in food, they are recommended to replace the enzyme modified starch.
Modern food processing has been developing technologies to improve the usefulness and safety of
food products of mass consumption without loss of quality characteristics of familiar consumer products.
Besides quality parameters regulated of standards, food must be safe in terms of microbial contamination that is regulated of sanitary laws [10] However, there are safety parameters, such as genetic, which are not spelled out for the products of mass consumption, but consumers are increasingly interested in this issue, especially in connection with the use of dietary supplements.
In connection with this work on the creation of new types of enzyme modified starches products are actual. Expansion of the range of meat products through the use of enzyme modified starches will contribute to the challenging task of creating of new forms of products which are not only safe for human health, but also have better functional state.
The aim is a comparative assessment of the impact of some modified and enzyme modified potato starches on the quality and biosafety of meat minced products .
Materials and methods Materials
Commercial grade of native potato starch was purchased at a local supermarket in Kazan, Russia. Fermented starches prepared using the commercial production of enzymes a-amylase (type II-A: from Bacillus sp., Sigma-Aldrich) and p-amylase (derived from barley, Fluka), freeze-dried enzymes. The investigations were potato starches: native, chemically modified (oxyamyl OPV-1, hydrolyzed, swelling on TU 9187-016-5747146-95, contributed Institute of Nutrition, Moscow) and enzyme modified.
Enzymatic hydrolysis
A flask equipped with a condenser and a mixer was used for the experiment. The solution with 30% (m/w) potato starch was preheated in a heating mantle until it reached the enzyme optimum temperature (below the gelatinization temperature of 60 °C). Fermented starches prepared using the commercial production of enzymes a-amylase (type II-A: from Bacillus sp., Sigma-Aldrich) and p-amylase (derived from barley, Fluka), freeze-dried enzymes. The concentration of enzymes is 2 ^g/g of starch. The pH of the mixture was controlled at enzyme optimum pH. After hydrolysis for 8 or 12 h, the enzyme was inactivated by adding H2SO4 to reduce the pH to below pH 4. Finally the starch was isolated by filtration, and then dried in a forced-air oven (40 °C) for 24 h. Enzyme modified starches produced by the action of a-amylase were identified 8-a and 12-a (8 and 12 hours fermentation time), starches, modified by p-amylase: 8-p and 12-p (8 and 12 hour fermentation time).
Preparation of experimental minced meat product
As experimental meat served chopped meat products - burgers, containing in its composition experimental and the native starches, as a control sample without the addition of starch (Tabl. 1). The beef, pork fat and onion mince twice for the preparation of the experimental samples. The salt, black pepper, water, potato starch (dry) added in cutlet meat. The cutlet mass was molded in the form of balls 100 g, which is heat treated for a couple for 20-25 minutes. Finished products were analyzed.
Table 1 - Formulation prototypes minced meat product containing potato starches introduced as components
Ingredients Composition content, g
Control Test
Beef 67,0 65,0
Fat, raw pork 8,94 8,94
Fresh onion 2,0 2,0
Ground black pepper 0,06 0,06
salt 1,2 1,2
starch - 2,0
water 20,8 20,8
semi-finished mass, g 100 100
Functional and technological methods
Water-holding capacity (WHC). The WHC of
samples was determined using a Carver press (Model C, Carver, Inc., Wabash, IN) using the method recommended by Wierbicki and Deatherage (1958) [11]. Before analysis, Whatman filter papers (#1) were stored overnight in a chamber above saturated KCl solution. Meat sample (0.5 g) was put on a filter paper, placed between two sheets of plexiglas, and compressed for 5 min at 500 psi. The inner circle area of meat film and the outer circle area of expressible juice were
measured with a planimeter. The WHC was expressed as the ratio of the meat film area to the expressible juice area [12].
Water-absorption capacity (WAC). A modified centrifugation method [13] was used. Five grams of sample were blended with excess distilled water (15 mL) for 1 min. The homogenized mixture was then poured and rinsed into a preweighed tube and centrifuged (Omnifuge RT, American Scientific Product, McGraw Park, IL) at 1980 rpm for 25 min. The remaining unabsorbed water was decanted after centrifugation and the water absorbed by meat was calculated. WAC was calculated as g H2O absorbed per g of meat.
Assay of cooking loss. Samples were weighed before and after cooking. Cooking loss was calculated as a weight difference between the heated and the unheated weight (%).
Moisture content was determined by Moisture Analyzer thermogravimetric MX-50 A&D (AND, Japan).
Assay of water-soluble protein. 3-4 g of ground sample was mixed with distilled water in the ratio 1:6 (by weight) and extraction carried out in the cold at 3-5 °C for 30 min. Then the precipitate was collected by filtration. The filtrate was used for the quantitative determination of water-soluble proteins. The amount of protein was determined using the technique of Bradford.
Assay of pigments. The conical flask with stoppered, wrapped in black paper for protection from light, placed 10 g sample [23]. Then 40 ml of chemically pure acetone (density 0.795), 2 ml of distilled water and 1 ml of hydrochloric acid were added. The contents of the flask was triturated until a paste-like slurry, followed by addition of the remaining solution. The flask was sealed and kept in a dark place for 1 hour, stirring occasionally, and then the contents of the flask was poured through a fluted filter into a conical flask. The filtrate (hematin hydrochloride in 80% acetone) contains pigments. The relative amount of the pigment was measured at 540 nm on a SF-2000 spectrophotometer (Sankt-Petersburg, Russia).
Assay of starch. In determining the content of free sugars and a starch, 1 g of crushed product sample was placed in flasks 1 and 2 to 100 ml. In 1 flask, for determination of total sugars and starch 50 ml of 0.5 % sulfuric acid was added and hydrolyzed starch in a boiling water bath for 15 minutes. Then 2 ml of a 30% solution of ZnSO4 and 15% K4[Fe (CN)6] 3H2O to clarify the solution were added and then the solution was adjusted to the mark with distilled water and filtered. In 2 flask free sugars were assay, 1 g of sample was mixed with 80 ml and extracted for 1 hour. The extract was clarified by adding 1 ml of ZnSO4 and K4[Fe (CN)6] 3H2O solutions then the solution was adjusted to the mark with distilled water and filtered. For each extract, 1 ml experimental extract was added to 3 ml of 9,10-dihydro-9-oxoanthracene (anthrone) under acidic conditions (0.2 g/100 mL of 76% sulfuric acid).
All tubes were vortexed quickly, and placed in boiling water bath for 10 minutes and then cooled in the dark for color formation (20 min). A blank was prepared with distilled water instead of sample extract. After boiling tubes solution was cooled to room temperature, the optical density at 610 nm against a control sample by a SF-2000 spectrophotometer (Sankt-Petersburg, Russia). A calibration curve was calculated in dextrose. In a first flask extract determined the total content of sugars and starches and on the second extract determined the content of free sugars.
Histological analysis
For histological analysis chopped products ready cut into pieces measuring 10*10x10 mm and heated in a 10% formalin solution for fixation for a week, and then washed of water. Cutting the samples was performed on a freezing microtome type. Preparation of slices produced at the luge microtome using microtome knives. The thickness of the slices obtained - 1, 2, 3, 4, 5, 6 microns. Straightened sections were placed on specially prepared glass slide. Glass degreased in alcohol-ether mixture (1:1). After receiving the slices were stained with hematoxylin according to standard procedure. Next cut remanded balm and covered with a cover glass, viewed under a microscope Axio Imager complete with a video camera, an increase of 400 times.
DNA-damaging activity
DNA-damaging activity was determined using strains of Escherichia coli: - Wp - wild type (all working repair system); three mutant strains defective in different ways repair: recA- - There is no process of post-replicative repair; polA- - synthesis of DNA-polymerase-1 is broken; uvrA- - excision repair is broken. The principle of the method consists in the selective inhibition of growth by the mutant E.coli strains compared to the wild type Wp [14].
Results and discussion
In this paper efficiency of the use of chemically modified and enzyme modified starches as prescription components in the production of meat minced products was analyzed. The concentration of starch was introduced 2% by weight of the feedstock, which corresponds to the allowable concentration (no more than 5%) the use of starches in the meat products, which involves the recommendation to their use.
The greatest WHC and WAC of chopped meat have been identified in the sample with oxyamyl OPB-1 and treated by a-amylase during 8 h starches (Tabl. 2). Enzyme modified starches were not inferior, and in some cases, and outscored of chemically modified starches.
The highest yield had model sample of minced products with oxyamyl and a-8 starches, the lowest output - products with swelling starch (Tabl. 2). Adding enzyme modified starches in recipes model samples led to increased yield of 5-14% compared with the products without starch. In case of fermented starches obtained
by prolonging of the fermentation time up to 12 hours (a-12 and p-12, starches,) the yield of the products was 82.3 and 80.7%, respectively
It is known that starch is introduced into the emulsion for increase of the viscosity of stuffing. The use of starch can not only increase the yield, but also the bioavailability of meat products. Modified starches are used as moisture- and fat-holding agents.
Moisture content is one of the most important indicators characterizing rheological properties of raw materials, and formed the organoleptic properties of the product and affects the yield.
Our data showed that the moisture content of model samples ranged between 59-68%, which is higher than in the control sample (Tabl. 2). The highest moisture content in the samples was with the introduction of a- 8 and oxyamyl starches, which correlates with the data on the WHC, WAC and cooking loss.
Table 2 - Functional and technological parameters of minced meat and chopped finished products with the introduction of chemically and enzyme modified potato starches
Variant of starch WHC, % WAC, % Moisture, % Cooki ng loss, %
control (w/o starch) 60 57 54 23,8
native 63 62 64 16,7
oxyamyl OPB-1 70 88 69 10,5
hydrolyzed 66 83 64 13,2
swelling 62 80 59 13,1
a-8 74 89 68 9,9
a-12 69 88 65 11,0
P-8 66 64 66 17,7
P-12 66 64 62 19,3
Native and modified starches have the ability to absorb up to 11 parts of water, however, retain water for a long time (during thermal processing and storage) can only modified starches. Meat products including modified starches are characterized by high richness and smaller mass loss products during heat treatment. However, during the heat treatment may be subjected as starches washout, and partial hydrolysis to form the free sugars are starch degradation products.
To determine the stability of chemically modified and fermented starches during the heat treatment was determined and the residual starch content of free sugars in the model samples. Despite the fact that the estimated amount of starch introduced should be 2%, the residual amount of starch in the model samples ranged between 0.5-1.7 g/100 g of product (Tabl. 3). Most starch concentration was in the samples with a-8, p-8 starches. Chemically modified starches have proved less resistant to thermal stresses in minced meat model system: residual starch content - 0.5-1.3 g/100 g of product. It is important, that the increase of the
time of starch fermentation (a-12 and P-12, starches) resulted to a decrease in their stability in the final product, which may be associated with a decrease in starch grains and enhancing their ability to leaching during thermal treatment.
The content of free sugars in the model sample products without starch in an amount of 0.014 g/100 g of product due to the natural transformation of glycogen to glucose when rigor mortis. Chemically modified starch were unstable and less stable starches by heat treatment as evidenced by the high levels of free sugars in the models with its introduction. Hydrolyzed starch was more stable under heat treatment.
Smallest content of free sugars were in the model samples with enzyme modified starches a-8 and p-8. Revealed an inverse correlation between the indicators of the amount of starch and glucose was revealed.
The results of determining the amount of soluble protein in model samples showed in Table 3. Product must contain components necessary for normal body metabolism, primarily needs replenishing human protein.
Table 3 - Physical and chemical properties of model samples of meat minced products made with chemically and enzyme modified starches
Variant of starch in meat products Concen tration of water-soluble protein, g/100 g product Relative amount of pigment, D, X=540 nm Starch concent ration, g/100 g of product Free sugar concent ration, g/100 g of product
control 0,2± 0,02 0,501± 0,002
(w/o starch) 0,036± 0,014±
native 0,33± 0,549± 1,254± 0,031±
0,03 0,005
oxyamyl OPB-1 0,39± 0,035 0,580± 0,003 1,076± 0,032±
hydrolyz ed 0,36± 0,025 0,576± 0,002 1,3 57± 0,035±
swelling 0,25± 0,025 0,472± 0,006 0,512± 0,038±
a-8 0,45± 0,592± 1,665± 0,036±
0,017 0,002
a- 12 0,42± 0,594± 0,899± 0,009±
0,012 0,003
P- 8 0,30± 0,556± 1,994± 0,029±
0,013 0,004
P- 12 0,28± 0,535± 0,841± 0,008±
0,014 0,005
The highest content of water-soluble proteins found in the model samples with a-8 and 12 starches With regards to the model samples made with the introduction of chemically modified starches, the highest content of water-soluble proteins found in the model with oxyamyl starch products (0.580 g/100 g) (Tabl. 3).
This trend correlates with the yield data, maximum content of water-soluble proteins corresponded to
samples having a maximum yield (oxyamyl, hydrolyzed and a-8 starches). Probably it is due to the retention of water-soluble proteins and high water-holding capacity of model meat with types of starches.
The content of pigments in meat products has an impact on indicators such as the color of the product, which is considered one of the most important criteria for the quality of products.
The maximum content of the pigments found in the model samples with a- 8, p-8 oxyamyl and hydrolyzed starches. The smallest amount of pigments contained in the model samples with swelling starch. The results suggest a positive effect of enzyme-treated starches used as prescription components, the color of the minced products.
Microstructural study was carried out only with the samples with the introduction of enzyme modified starches. This made it possible to analyze the distribution of starches between the components of minced meat.
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Fig. 1 - Image of light microscope of histological sections of meat minced products prepared with enzyme modified starch (A - Control (w/o starch), B - native, C - a-8 starch, D - a-12 starch, E - 08 starch, F - 0-12 starch)
Minced meat samples formed from fragments of muscle, connective and adipose tissue (Fig. 1). The presence of microstructural changes is caused of thermal effects in muscle tissue. Adipose tissue is represented by fragments of cells of various sizes and fat droplets dispersed sufficiently strong interfiber spaces and infiltrating fragments and pieces of meat. Starch particles located between minced meat
colored by histological dyes with a fairly clear spatial boundaries.
As a result of histological studies of model samples was clearly demonstrated in a spatial arrangement made of starches in the thick stuffing. Native starch was visible like clear granules in the structure, however polysaccharide unevenly located in the structure of the cut, revealed his flock. In case of a-8 and P-8 starch, granules settles down in muscular space. The use of a-12 and P-12 led to the sealing of structure, despite the uniformity of their location in the structure. These may adversely affect the consistency of the finished products.
The question of how can influence various synthetic additives including starches, and modified, the genetic material of the person is currently relevant.
In connection with this model genotoxicity of product samples was studied. Water extracts modeling products with the introduction of chemically modified starches showed DNA-damaging effect on all or two strains E.coli (Tabl. 4).
The water-soluble components with the introduction of a model sample of native starch did not possess of DNA damaging activity (95% survival index). The water-soluble components of model samples of with a-8, a-12 and P-12 starches showed no DNA-damaging properties against the defective strains (survival index of 100% or more).
DNA-damaging activity were determined of aceton-soluble components. Unlike water-soluble components, aceton-soluble components had greater genotoxic potential.
Table 4 - DNA-damaging activity of water-soluble and aceton-soluble components of model samples of meat minced products made with chemically and enzyme modified starches
Variant of starch in meat products Genotoxicity, survival index, %
water-soluble aceton-soluble
Rec A- Uvr A- Pol A- Rec A- Uvr A- Pol A-
control (w/o starch) 106 89 99 91 78 89
native 95 96 98 94 90 90
oxyamyl OPB-1 94 92 86 91 93 95
hydroly zed 113 104 101 108 95 94
swelling 90 91 89 100 102 94
a- 8 100 96 101 93 93 98
a- 12 117 125 114 91 89 92
P- 8 92 101 103 101 88 99
P- 12 117 122 101 96 92 91
The acetone-soluble fraction of model samples with fermented starches showed no genotoxic effect (in all cases the survival index was greater than 95%).
Thus, the model samples of chopped meat products from fermented starches for functional and technological and physico-chemical parameters are not inferior model samples with chemically modified starches. In terms of biosafety and environmental use of fermented starches in the production of meat minced products preferable as revealed lower levels of the genotoxicity of water-soluble and aceton-soluble fractions of model products.
Except it was shown that a-8 and P-8 potato starches possess high resistance to action a-amylase that is positive from the point of view of decrease of caloric content of a meat food [15]. Summarizing, we can say that in terms of functional and technological, physical, chemical, biological and histological properties of the application of a-8 and P-8 potato starches in the technology of meat minced products is provided of biologically safe food production.
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© E. V. Nikitina - PhD, associate Professor of Technology of food production KNITU, REC "Pharmaceuticals KFU, ev-nikitina@inbox.ru, L. Z. Gabdukaeva - Ph.D., assistant Professor of Technology of food production KNRTU, carramba@bk.ru; M. S. Egkova -doctor of veterinary Sciences, Professor of department of Technology of meat and milk production KNRTU.
© Е. В. Никитина - канд. биол. наук, доц. каф. технологии пищевых производств КНИТУ, ev-nikitina@inbox.ru; Л. З. Габдукаева - асп. той же кафедры, carramba @ bk.ru; М. С. Ежкова - доктор ветеринарных наук, профессор каф. технологии мясных и молочных производств КНИТУ.
