METHODS OF DETERMINATION OF PARAMETERS OF FERMENTED WHEY-MALTY MIXTURES

The article presents main methods of studying restored whey-malty mixtures after fermentation by lactose-fermenting yeast and saccharomyces for getting a beverage of the kvass type. Reports on research projects (2018), «EUREKA: Life Sciences» Number 5


Introduction
It is expedient to introduce technologies of products, based on whey, that don't need essential technical re-equipping of an enterprise and establishing of new lines, in production [1,2].One of simplest technological ways of processing whey is beverage production.
Whey is a product with a natural set of nutritive and biologically full-value components.The qualitative composition and number of macro-and microelements of whey products essentially exceed traditional refreshing beverages.Whey contains all irreplaceable amino acids.90 % of carbohydrates are presented by disaccharide -lactose.The content of glucose in whey of sour-milk cheese -0,7…1,8 %.It is conditioned by hydrolysis of lactose at processing.Among other carbohydrates, arabinose, lactulose and amyloid are present [3].Fat-and water-soluble milk vitamins are transformed in whey; the latter ones almost completely.At the same time cheese whey contains much more of them than one of sour-milk cheese.
Dry whey contains almost all milk components and has the low energetic value, can be considerably used for manufacturing products with the combined composition, for example, icecream, yogurt, confectionary products and so on.The high biological value of whey is conditioned by protein substances, and also vitamins, hormones, organic acids, immune bodies, microelements.Dry whey on the organoleptic level is combined with different vegetable ingredients, for example, wheat sifting, cellulose, extrudates and grain malts and so on.It is possible to use dry rye malt with mass share (8,0±0,25) % in mixtures with dry whey [6].Solubility is an important parameter for using dry whey in the composition of such substances for restoring.This parameter for dry whey doesn't exceed 0,8 cm 3 of a damp residue [7].The existent method of determining this parameter is general and suitable for the objective assessment of dry malt [8,9].
Fermented beverages, based on whey, of the kvass type combines value components of the main raw material and products of metabolism of microorganisms, created at fermentation (ethyl alcohol, volatile acids, enzymes, different aromatic compounds and so on) [10].
All types of whey beverages have limited storage terms.Prolongation of suitableness and convenience of using this assortment both in production conditions and for the wide circle of con-Food Science and Technology sumers needs development of correspondent technologies.Malt with rusks is contained by recipes of dry concentrates of kvass for restoring in water [11].The use of dry concentrates, based on whey and rye malt, as a base of kvass is possible.
Problems that need a scientific substantiation include identification of such side fermentation products as higher alcohols (propyl, isoamyl, isobutyl and so on) with a typical smell and ability to create esters that essentially influence a fragrance of a fermented beverage [12,13].
The aim of the work is determination of effective methods of studying restored whey-malty mixtures after fermentation for developing dry concentrates and selection of yeast that allows to improve the technology of fermented beverages.

Materials and Methods
Whey-malty mixtures were prepared on the base of dry whey and rye malt.Physical-chemical parameters of dry whey, according to SSU 4552:2006 (European analogue -«Sweet whey powder» ISO 9001; ISO 14001; FSSC 22000) are presented in Table 1.Sodium hydroxide with concentration 0,1 mol/dm 3 for 100 g of the dry substance of malt, cm 3 35±1,75 Iodine solution with concentration 1 mol/dm 3 for 100 g of the dry substance of malt, cm 3 17±0,85 At the first stage of the experimental studies there were prepared the dry mixtures with different ratios of malt and whey 1:1,5; 1:2; 1,5:1; 2:1 by mixing.The choice of proportions was conditioned by organoleptic parameters of the restored mixtures.At the second stage there was produced whey-malty wort with introducing dry substance 10 %, restored at temperature 35…45 °С, intensively mixing, the temperature was increased to 75...80 °С for transforming extract substances into the solution (reducing sugars, soluble pentosans, nitrogen-containing compounds and so on).Then the mixture, cooled to 25…30 °С, was set for decanting for eliminating residues of denatured proteins of whey to the malt residue.For malt fermentation there were used lacto-fermenting yeast Kluyveromyces lactis 469 with amount 40 mln cell/cm 3 of wort.The effectiveness of aforesaid yeast in whey was proved by the previous studies [14,15].Fermentation was realized to temperature 30 °С during 36 hours.

Food Science and Technology
Yeast cultures, used in the work, were received from the "Collection of strains of microorganisms and lines of plants for food and agricultural biotechnology" SI "Institute of food biotechnology and genomics" NAS of Ukraine.
Correspondent races of lactose-fermenting yeast and saccharomyces were selected by methods that objectively estimate the effectiveness of fermentation.
The physiological condition of yeast was estimated by the general number of cells with glycogen with coloration by Lugol solution.The amount of yeast in 1 cm 3 was determined by the direct calculation in Goryaev chamber [16][17][18].
According to the literary data [19], normal yeast contains no more 10 % of dead cells.The high content of such cells causes decelerated fermentation, favors development of side microflora and autolysis of yeast.For functioning normally, yeast must contain 70…75 % of cells with glycogen.Less number characterizes insufficient nutrition of yeast, starvation.Such yeast multiply slower, lag-phase is longer, fermentation process is decelerated.
The following stage was the study of fermentation activity of different yeast races in wheymalty wort and suitableness for getting fermentative beverages with the standardized content of ethyl alcohol.The raw material -is a restored mixture of dry whey and rye fermented malt.
The control parameters of yeast biochemical activity were the amount of accumulated ethyl alcohol, depth of sugars utilization in the process of cultivation by the content of reducing substances for the final term of fermentation.The research objects were selected as yeast Saccharomyces casei, Saccharomyces cerevisiae M-5, Kluyveromyces lactis 2452, Kluyveromyces lactis 469, Saccharomyces lactis 95.The control -wort, based on water and malt, produced according to the classic technology using yeast Saccharomyces cerevisiae-P-87.
Then there was estimated the ability to development in different races of yeast in fermented whey-malt wort.For comparing the ability to accumulation at different variations, there was selected the monoculture Saccharomyces cerevisiae Р-87 [16].Such yeast is not able to metabolize lactose independently or to stimulate the development of lactose-fermenting yeast.There was also studied the consistent cultivation of Saccharomyces cerevisiae Р-87 with lactose-fermenting yeast Saccharomyces lactis 95 on the nutritive medium -restored mixture of whey and malt.During the experiment there was determined the number of yeast cells at fermenting wort of the restored mixture (ratio of malt to whey-1:2).The process was fixed during 36 hours.
Specification of the temperature of wort fermentation was realized in samples, cultivated by different yeast races at temperatures 24…36 °С with interval 2 °С.
At the fermentation process the amount of emitted carbon dioxide was controlled by the weight method.Mature mash was distilled for determining the mass share of alcohol in the distillate.The image of the laboratory device for extracting ethyl alcohol is presented on Fig. 1.The content of reducing substances in whey-malty wort was determined by the iodometric method (this index before fermentation was 5,24 %).The main parameter of the intensity of this process is the amount of emitted carbonic acid in the time unit, so fermentation continued up to the stop of emission of carbon dioxide [16].
Identification of side fermentation products of fermented whey-malty wort.
The characteristic of the smell of higher alcohols and esters depending on construction is presented in Table 3 [12].

Fusel tone
Identification of side fermentation products in the distillate of fermented whey-malty wort was realized by the gasochromatographic method by determining higher alcohols С 1 -С 5 [20].Conditions of the chromatographic analysis of determination of volatile substances are presented in Table 4.

Conditions of analysis Value
Chromatography The image of the laboratory equipment Chrom 5 (plant "Laboratory devices" Czechia) for the gasochromatographic analysis is presented on Fig. 2.

Fig. 2. Gasochromatographic equipment Chrom 5
Determination of quantitative ratios of higher alcohols, aldehydes and esters in the distillates was realized by the method of internal normalization using "Chromprocessor" software [21].

Еxperimental procedures
The results of the experimental studies of accumulation of yeast cells Kluyveromyces lactis 469 and their percent with glycogen from the general concentration at fermentation of wort of the restored mixture at different ratios of dry malt and whey are presented in Table 5.The most increment of cells Kluyveromyces lactis 469 is observed in the restored mixture of malt to whey as 1:1,5 and 1:2 from 4 to 16 hour of fermentation.Indices are an evidence of the effectiveness of utilization of lactose by yeast.
The increment of cells with glycogen (table 5) was more intensive in samples with the other ratio of malt to whey -1,5:1 and 2:1.Thus, at 36 hour of fermentation the general amount of yeast Kluyveromyces lactis 469 was 67,2 and 68,9 % respectively.
Taking into account the fact that the concentration of yeast cells at 36 hour of fermentation was maximal at level 74,9 mln/cm 3 , wort of the dry mixture with malt: whey ratio as 1:2 was selected for studying fermentation activity.
Specification of the temperature of wort fermentation was realized in samples, cultivated by different yeast races at temperatures 24…36 °С with interval 2 °С, fermentation duration 36 hours.The initial concentration of yeast cells in all samples was 40 mln/cm 3 of wort.The dependence of accumulation of yeast cells on the cultivation temperature is presented on Fig. 3.

Fig. 3. Dependence of accumulation of yeast cells on the cultivation temperature
The data of fig. 3 testify that the active growth of yeast cells of all races takes place at the temperature from 28 to 32 °С.For Kluyveromyces lactis 469 the maximal yeast accumulation (67…69 mln cell/cm 3 of wort) is observed at fermentation temperature -30…32 °С, for Saccharomyces cerevisiae Р-87 and Saccharomyces cerevisiae M-5 -30…34 °С.
According to the results, in further studies by the aforesaid methodologies it is possible to use lactose fermenting microorganisms and saccharomyces, although lactose fermenting yeast demonstrated the lower result.Their dynamics of biomass accumulation is enough, so it is expedient to use them for fermentation of restored whey-malt wort.According to generative capacity, the following yeast races were selected for fermentation: Saccharomyces cerevisiae Р-87, Kluyveromyces lactis 469 and Saccharomyces lactis 95.As to the studies by the gaschromatographic method of side fermentation products of wheymalt wort, fermented by Saccharomyces cerevisiae Р-87, there were fixed values of methyl acetate concentration at level (11,72±0,59) mg/dm 3 and ethyla cetate (92,17±4,61) mg/dm 3 .Such indices are optimal for forming a harmonious smell [9].According to organoleptic parameters, wort is an opaque dense liquid of the dark-brown color, sour-sweet, malty taste without an expressed bitterness.Its smell is harmonious malty with fruit and flower tones.
Wort, fermented by yeast Kluyveromyces lactis 469, has the analogous taste, but with the more expressed bitterness.There are observed lower concentrations of methyl acetate (8,03±0,40) mg/dm 3 ) comparing with the limit one, ethyl acetate ones are essentially higher.At the same type there is observed the malty smell with weakly expressed fruit and sharp sour tones.

Conclusions
Determination of fermentative activity of yeast by the concentration of yeast cells (mln/cm 3 ) and their percent with glycogen of the general number is effective at selecting yeast races for fermenting restored whey-malty mixtures.
The results of the gasochromatographic studies of determining side fermentation products of whey-malty wort allows to estimate their qualitative composition objectively as a result of the effect of Saccharomyces cerevisiae Р-87.Presence of this yeast race in the nutritive medium positively influences metabolism of a producer, stimulating biosynthesis or transformation of aromatic substances of the nutritive medium.The use of such lactose fermenting yeast for fermentation at temperature 30...34 ºС of restored whey-malty wort indicate objective possibilities for the effective use in production systems.
The given information is recommended to be used for substantiating parameters in the technology of fermented whey-malty beverages of the kvass type.

Table 1
Physical-chemical parameters of dry whey

Table 3
Characteristic of smells of alcohols depending on molecular construction

Table 5
Dynamics of accumulation of yeast cells and their percent with glycogen from the general concentration