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Оцтка лактозозброджувальних дрiжджiв для ферментаци сумiшi сироватки та яблуч-ного пектину в клтковиш дозволила обрати Kluyveromyces lactis 868-К як найбшьш ефектив-т для одержання ферментованих напогв високих ароматичних властивостей. Встановлена рациональна температура гх ферментаци 30...32 °С, при якш спостеркаеться максимальне накопи-чення дрiжджових клтин 70,5...71,2 млн/см3 та етилового спирту 0,64...0,69 % об.
Ключовi слова: яблучний пектин в клтковит, молочна сироватка, лактозброджуючi дрiжджi,
ферментований натй, ароматичш компоненти □-□
Оценка лактоферментирующих дрожжей для смеси сыворотки и яблочного пектина в клетчатке позволила выбрать Kluyveromyces lactis 868-К как наиболее эффективные для получения ферментированного напитка с высокими ароматическими свойствами. Установлена их рациональная температура ферментации 30...32 °С, при которой наблюдается максимальное накопление дрожжевых клеток 70,5...71,2 млн/см3 и этилового спирта 0,64...0,69 % об.
Ключевые слова: яблочный пектин в клетчатке, молочная сыворотка, ферментированный
напиток, ароматические компоненты -□ □-
UDC 663.86:637.344
|DOI: 10.15587/1729-4061.2016.596921
STUDY OF LACTOSE-FERMENTING YEASTS KLUYVEROMYCES LACTIS FOR WHEY AND APPLE PECTIN MIXTURE FERMENTATION
O. Grek
PhD, Assistant Professor* E-mail: grek.nupt@mail.ru N. C h e p el
PhD, Assistant Professor* E-mail: natachepel@yandex.ru O. Krasulya
PhD, Assistant Professor* E-mail: olena_krasulya@ukr.net *Department of technology of milk and dairy product National University of Food Technology Volodimirskaya str., 68, Kyiv, Ukraine, 03680
1. Introduction
Nowadays simultaneous providing of saving resource technologies of high-quality dairy products with the mul-ticomponent compositions is relevant. In addition, there is a problem of whey utilization because its volumes are growing steadily in order with the recycling for milk protein concentrates [1]. Thus, about nine liters of whey are obtained by producing each kilogram of cheese and the world whey production amounts are about 82 million metric tons [2, 3].
The prospects for increasing the utilization of secondary raw materials are closely related to the production of whey products which include not only whey but also various non-dairy functional components [4]. Despite the regulatory framework of dairy products based on whey technologies existence, the process of their implementation has its difficulties [5]. There is a number of whey processing technologies using bar membrane methods and biotransformation into carbohydrate and nitrogen-containing derivatives but the processes require special equipment [6-9]. The most economically expedient is the industrial realization of fermented whey beverage technologies which can be produced on any dairy enterprise equipment and does not require additional investment or energy [10-12].
2. Analysis of published data and problem statement
The fermented whey beverages contain valuable components of whey and secondary metabolic products obtained
during fermentation such as proteins, lactic acid, ethanol, enzymes and volatile compounds [13].
Whey has a high biological value due to the amino acid content (isoleucine, leucine and valine) that stimulate specific intracellular metabolism pathways associated with protein synthesis and play a role in the hormonal response to feeding as the stimulator of insulin secretion [14]. But whey is not a balanced source of nutrients and contains high lactose content [15]. The whey fermentation is initiated by lactose hydrolysis by lactic acid bacteria (LAB). During fermentation, around 23-30 % of lactose is transformed into lactic acid. LAB do not metabolize lactose directly but by using lactose permease, it is transferred into the cells where it is hydrolyzed to glucose and galactose [16]. Some humans do not possess sufficient amounts of p-D-galactosidase in their digestive system and, therefore, are not able to digest lactose. This undigested lactose causes problems like bowel cramps and diarrhea.
In this aspect special attention is being paid to the development of fermented whey beverage technologies using the fermentation of whey and functional component mixture by lactose-fermenting yeasts. The main requirements of their production are the choice of appropriate ratio of whey and functional components, suitable starter cultures of yeasts and investigation of the fermentation according to the biochemical activity of lactose-fermenting strains.
Quality properties, the nutritional value of whey beverages can be improved by adding hydrocolloids as a functional component [17]. In our previous work, the possibility of using apple pectin in the fiber (APF) added in whey beverage technologies was shown [18]. This hydrocolloid consists
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of wheat bran, apple powder and pectin, and increases the viscosity of whey. The wheat bran provides dietary fibers which modulate hunger and satiety moods, influence the glycemic, lipid and inflammatory status of consumers, and have prebiotic activity [19]. The apple powder is a rich source of carbohydrates, minerals, phenolic acids and flavonoids; it also shows high values of hydration properties such as water holding, water retention and swelling capacity as well as health-enhancing benefits such as cancer cell proliferation, lipid oxidation decrease and cholesterol lowering [20]. The pectin can reduce cholesterol, delay gastric emptying, and induce apoptosis of colon cancer cells [21].
The enzyme (P-D-galactosidase) which is used to hy-drolyze the lactose in milk and dairy products is produced from yeasts Kluyveromyces spp. [22]. Kluyveromyces lactis included lactose-fermenting yeast strains which produce P-galactosidase that possesses the ability to assimilate lactose when lactose is used as a carbon source [23].
3. Aim and objectives of the study
The main aim of this work was to evaluate some lactose-fermenting yeast Kluyveromyces lactis strains and choose the cultivation conditions in order to obtain the most effective fermentation process of whey and apple pectin in the fiber (APF) mixture.
According to this aim, the following research objectives have been identified:
1. Study of exponential multiplication phase of lactose-fermenting Kluyveromyces lactis strains.
2. Yield of fermentation and rate of fermentation.
3. Evaluation of odor-active compounds in whey and APF fermented beverages.
4. Research materials
4. 1. Whey and apple pectin in the fiber (APF) mixture
The whey from cottage cheese was kindly supplied by Joint-Stock Company "Pyriatynsky dairy plant" (Pyriatyn, Poltava Oblast, Ukraine). APF was purchased from Private Business Health Food Factory "Nashe Nasledie" (Donetsk, Ukraine). APF consists of wheat bran (60 %), apple powder (38 %) and pectin (2 %). It had the following physical properties: 33.0±0.99 % solubility, 90.0±2.70 % moisture-retaining power in the water and 84.0±2.52 % moisture-retaining power in the whey.
It was formulation of a fermentation medium based on whey and APF mixture, with the following initial properties: 6.5±0.03 % dry matter, 4.6±0.02 % lactose, 1.3±0.01 % protein, pH 4.5.
4. 2. Yeast strains
Ten lactose-fermenting yeast Kluyveromyces lactis strains, coded 42 K, 95, 300, 304, 317, 318, 325, 469, 868-K and 2452 were kindly donated from the Culture Collection of Microorganisms and Plant Lines for Food and Agricultural Biotechnology of Institute of Food Biotechnology and Genomics National Academy of Science of Ukraine.
4. 3. Preparation of fermentation medium
The fermentation medium containing whey and APF mixture was prepared by the following procedures. 20 g of
APF were mixed with 180 g of whey at a temperature of 35±5 °C for 10-15 min. The medium was then heated to a temperature of 74±2 °C for 15-20 seconds and cooled to a temperature of 30±2 °C and stored in a refrigerator at a temperature of 5 °C.
The obtained medium had the following initial properties: 8.5±0.04 % dry matter, 4.5±0.02 % reducing substances and pH 4.6.
4. 4. Preparation of the inoculum
For obtaining the inoculum, the yeasts during cultivation in aerobe conditions were grown on the streaked plates on whey and APF mixture liquid medium at a temperature of 30±2 C for 24 h. Cells of cultures in the concentration of 1106 CFU/ml of the medium were transferred for inoculation of 2 l flasks with 1 l of whey and APF mixture, liquid medium. Fermentation in flasks was performed at a temperature of 30±2 C in a rotary shaker (220 rpm) for 24 h. Biomass was separated from the culture broth by filtration on the vacuum filter.
4. 5. Preparation of samples of whey and APF fermented beverages
1 g of wet biomass culture of each starter was added to 200 ml fermentation medium. All samples were then placed in a stationary condition in a heating chamber with the air temperature of 24 °C, 26 °C, 28 °C, 30 °C, 32 °C and 36 °C for 6±0.5, 12±0.5, 18±0.5, 24±0.5, 30±0.5 and 36±0.5 hours. Fermentation was stopped by rapid cooling to a temperature of 4±2 °C.
The samples of fermented whey without adding of APF were served as a control.
5. Research methods
The evaluation of lactose-fermenting Kluyveromyces lactis strains for whey and APF mixture fermentation was performed according to yeast cell concentration during fermentation, pH, CO2 content, the content of dry matter and reducing substances, ethanol content in whey and APF fermented beverages and volatile compounds determination in distillate products.
5. 1. Yeast cell concentration determination
The yeast cell concentration (106 CFU/ml) was estimated by direct counting in Goryaev's chamber with Lugol solution embellishment. All parameters were evaluated in every 2 h of fermentation. All samples of fermented whey and APF beverages were prepared and measured three times.
5. 2. Ethanol content determination
The products had undergone distillation with obtaining their distillate products. The ethanol content of distillate products was determined at a temperature of 24 °C, 26 °C, 28 °C, 30 °C, 32 °C and 36 °C for 6±0.5, 12±0.5, 18±0.5, 24±0.5, 30±0.5 and 36±0.5 hours using the method 984.14 AOAC (AOAC, Washington, USA).
5. 3. Determination of odor-active compounds by gas chromatography-olfactometry
Gas chromatography-olfactometry (GC-O) was performed using an Agilent 7890A series gas chromatograph (Agilent, Palo Alto, CA, USA) equipped with flame ioniza-
tion detector (FID), sniffing port ODP3 (Agilent, Palo Alto, CA, USA), and a DB - 5MS capillary column (30 m length x0.25 mm i. d. *0.25 |im film thickness, J&W Scientific, Folsom, CA, USA). The GC temperatures were controlled as follows: 40 °C (5 min); 4 °C/min to 120 °C; 16 °C/min to 220 °C (10 min). One |L of the sample of distillate products of whey and APF fermented beverages was injected in splitless mode and GC effluents were split to an FID and a sniffing port (1:1 ratio). The carrier gas was helium at a constant flow rate of 0.8 mL/min. The injector and detector temperatures were 230 °C and 250 °C, respectively.
The identification of volatile compounds including odor-active compounds was positively confirmed by comparing their retention times with those of authentic standard compounds. The retention indices (RI) values of volatile components were calculated with C1-C5 higher alcohols as external standards.
For quantitative analysis, the relative concentrations of odor-active compounds in 2 types of samples were analyzed by the CHROMPROCESSOR computer package (Avangard TM, Ukraine; 2008). The recovery % for each odor-active compound was obtained by the addition of the mixed solution containing 8 standard compounds (w/v, 100 |g/mL). Concentrations of odor-active compounds were calculated on the base of calibration and recovery.
5. 4. Statistical analysis
All data are expressed as mean values ± standard deviation (SD). Statistical differences between experimental groups were assessed by analysis of variance (ANOVA), using the COSTAT software package (Cohort Software, CA, USA). The main values were compared with LSD test (P<0.05).
6. Research results and discussion
6. 1. Study of exponential multiplication phase of lactose-fermenting Kluyveromyces lactis strains
The exponential multiplication phase of lactose-fermenting Kluyveromyces lactis strains shows the influence of the fermentation medium on their growth and reproduction. Above presented data of exponential multiplication phase of lactose-fermenting Kluyveromyces lactis strains are system-ized in Table 1.
During cultivation in aerobic conditions the biomass yield was the highest by yeast cultivation in whey and apple pectin in the fiber mixture with Kluyveromyces lactis 868-K strain (71.3*106 CFU/ml). Conversely, Kluyveromyces lactis 300 strain showed the lowest fermentation potential. The yeast cell concentration has not been changed significantly for Kluyveromyces lactis 300 fermentation comparing with the inoculum and such fermentation was considered to be ineffective. Other lactose-fermenting yeast cell concentrations increased by 33-70 % after 24 h of fermentation compared with the inoculum.
During the alcoholic fermentation, the dynamics of CO2 accumulation is positively correlated with the dynamics of biomass accumulation. Kluyveromyces lactis 868-K strain performed the most effective fermentation among 10 investigated lactose-fermenting strains. It accumulated the highest yeast cell concentration and CO2 content, and the lowest reducing substance content at the end of fermentation.
Thus, CO2 content was six times lower and reducing substances content was 3.6 times higher for Kluyveromyces lactis 300 strain than for Kluyveromyces lactis 868-K strain. pH values and dry matter contents have not been changed significantly after fermentation for 50 % of tested yeast strains (Kluyveromyces lactis stains coded 300, 304, 317, 318, 326). For other 50 % of tested yeast strains (Kluyveromyces lactis strain coded 42-K, 95, 868- K, 2452) pH values have been decreased from initial 4.60 to 3.50-3.85 and dry matter contents have been declined from 8.5 % to 6.3-7.5 %. The reducing substance content diminution was more considerable than dry matter content and made up 7.6-27.6 % from initial.
6. 2. Yield of fermentation and rate of fermentation
The exponential multiplication phase of Kluyveromyces lactis 868-K strain growth was recorded between the 6th and the 24th h of whey and APF mixture fermentation (Fig. 1, a). Maximum yeast cell concentration was observed on the 30 h of whey and APF mixture fermentation and control. But this parameter of whey and APF mixture fermentation was 1.59 % less than for the control (71.3*106 and 72.5*106 CFU/ml in whey and APF mixture fermentation and control, respectively). The yeast cell growth decrease for the mixture compared with the control can be associated with the presence of unfermentable dietary fiber in APF which decreases substrate accessibility.
Table 1
Physical, chemical and microbiological properties of fermented beverages based on whey and APF (n=3)
Lactose-fermenting yeast strains Yeast cell concentration, (106 CFU/ml) PH CO2 content, g/100 ml Dry matter content, % Reducing substance content, %
Kluyveromyces lactis 42 K 65.6a(±0.09) 3.60a(±0.00) 1.28a(±0.00) 6.4a(±0.01) 0.40a(±0.00)
Kluyveromyces lactis 95 67.6a(±0.10) 3.80a(±0.00) 1.20a(±0.00) 6.4a(±0.01) 0.47b(±0.00)
Kluyveromyces lactis 300 45.2b(±0.17) 4.20b(±0.01) 0.24b(±0.00) 8.1b(±0.03) 1.24c(±0.02)
Kluyveromyces lactis 304 55.8c(±1.11) 4.40b(±0.01) 0.30c(±0.00) 8.1b(±0.03) 0.69d(±0.00)
Kluyveromyces lactis 317 60.8d(±0.36) 4.40b(±0.01) 0.85d(±0.00) 8.0b(±0.03) 0.82e(±0.01)
Kluyveromyces lactis 318 62.3d(±0.36) 4.40b(±0.02) 0.96e(±0.01) 8.0b(±0.03) 0.64d(±0.00)
Kluyveromyces lactis 325 58.6d(±0.34) 4.30b(±0.02) 0.79d(±0.00) 8.0b(±0.03) 0.68d(±0.00)
Kluyveromyces lactis 469 63.6d(±0.37) 3.85a(±0.01) 0.86d(±0.00) 6.5a(±0.00) 0.74f(±0.00)
Kluyveromyces lactis 868-K 71.3e(±1.31) 3.70a(±0.01) 1.45f(±0.01) 6.3a(±0.00) 0.34g(±0.01)
Kluyveromyces lactis 2452 62.9d(±0.37) 3.50a(±0.01) 0.68g(±0.02) 7.5b(±0.02) 0.63d(±0.00)
Lsd0.05 0.2290 0.0021 0.0003 0.0029 0.0006
SE± 0.189 0.00 0.00 0.00 0.00
Note: a-h Mean ±SD values having the same superscript within a column are insignificantly different (P<0.05)
b
Fig. 1. Rate of fermentation of the medium based on whey and APF from Kluyveromyces lactis 868—K strain: a — yeast cell concentration; b — CO2 content
Presented data of the rate of fermentation of the medium based on whey and APF from Kluyveromyces lactis 868-K strain are concordant with other investigation where Kluyveromyces lactis maximum biomass yield and p-galacto-sidase activity are achieved after 28 h of fermentation [23].
Maximum CO2 production was observed after 30 h of fermentation (1. 2 and 1.45 g/100 ml in whey and APF mixture fermentation and control, respectively) (Fig. 1, b).
Yeast cell concentration didn't vary significantly at temperatures ranging from 28 to 36 °C (Fig. 2, а) in whey and APF mixture fermentation and control. The highest ethanol content (Fig. 2, b) was registered at a temperature of 32 °C in whey and APF fermented beverages and control (0.69 and 1.02 %, respectively).
According to the regulatory requirements for soft drinks (Ukraine State Standard 4069:2002) ethanol content allowed in the product is not more than 1.2 % vol. [24]. Therefore obtained results can be used to implement the technology of whey and APF fermented beverages.
6. 3. Evaluation of odor-active compounds in whey and APF fermented beverages
A total of 8 odor-active compounds were found in whey and APF fermented beverages such as acetaldehyde, meth-ylacetate, ethylacetate, methanol, n-propane, isobutane, 2-methyl-1-butanol, 3-methyl-1-butanol. Table 2 lists their retention indices, odor descriptions, and aroma threshold values (ATVs).
Table 2
Gas chromatography-olfactometry properties of odor-active compounds in whey and APF fermented beverages
No. RIa Components Odor descriptions ATVb
1 525 Acetaldehyde floral, sweet 50 - 100
2 596 Methylacetate fruit, floral 30
3 607 Ethylacetate fruit essence 180-200
4 624 Methanol fermented, malt 20
5 719 n-propane vegetable, onion 100-500
6 725 Isobutane floral, rose 100-200
7 737 2-methyl-1-butanol fermented, malt, wine 300
8 739 3-methyl-1-butanol fermented, malt, wine 300
Note: RIa; retention indices on DB-5MS column were determined using C1-C5 higher alcohols as external standards; ATVb; aroma threshold values of odor-active compounds in water (ppb) [25, 26]
The unique sensory properties of different types of fermented beverages often are due to minor differences among the volatile compounds present. By using instrumental methods for qualitative or quantitative evaluations of these differences, in addition to sensory techniques, quality assurance analysts can obtain a wealth of information about their products [28]. Differences of odor-active compounds of whey and APF fermented beverages showed the pathway of biochemical processes in whey and APF mixture fermentation and indicated the biochemical activity of lactose-fermenting yeasts Kluyveromyces lactis. Table 3 lists contents of odor-active compounds in whey and APF fermented beverages and control with ten lactose-fermenting yeasts Kluyveromyces lactis.
Fermentation temperature, °C Fermentation temperature, °C
a b
Fig. 2. Dependence of whey and APF mixture fermentation temperature on: a — yeast cell concentration; b — ethanol content
Table 3
Contents of odor-active compounds in whey and APF fermented beverages and control (n=3)
Lactose- Concentrations of odor-active compounds in whey and APF fermented beverages / control*, mg/dm3
fermenting yeasts Acetaldehyde Methylace-tate Ethylacetate Methanol n-propane Isobutane 2-meth-yl-1-butanol 3-meth-yl-1-butanol
169.41a 7.69a 186.12b 20.35a 127.53f 271.88e 34.89a 17.92a
Kluyveromyces lactis 42 K (±0.25)/ 204.98a* (±0.01)/ 9.31b* (±0.70)/ 225.20f* (±0.03)/ 24.62d* (±0.98)/ 154.31a* (±5.00)/ 328.79h* (±0.05)/ 42.21b* (±0.02)/ 21.68d*
(±0.31) (±0.03) (±1.73) (±0.14) (±0.23) (±0.46) (±0.16) (±0.12)
229.04a 6.69d 195.61d 19.73b 137.53b 261.80f 20.78a 16.24b
Kluyveromyces lactis 95 (±0.34)/ 267.97b* (±0.03)/ 7.82h* (±1.15)/ 228.83f* (±0.07)/ 23.08a* (±0.52)/ 160.91a* (±2.01)/ 306.31b* (±0.03)/ 24.31d* (±0.06)/ 19.00e*
(±0.40) (±0.01) (±1.76) (±0.03) (±0.24) (±1.16) (±0.14) (±0.34)
164.62d 8.89b 498.17f 69.48c 133.09f 307.55f 60.72a 18.54a
Kluyveromyces lactis 300 (±0.97)/ 177.79a* (±0.03)/ 9.61d* (±3.83)/ 538.02a* (±1.38)/ 75.03e* (±1.02)/ 143.73f* (±2.38)/ 332.15d* (±0.09)/ 65.57b* (±0.02)/ 20.02d*
(±0.27) (±0.06) (±0.81) (±1.38) (±1.11) (±1.96) (±0.24) (±0.11)
157.80c 8.12b 473.18b 74.99h 127.32c 278.44a 54.56h 18.89b
Kluyveromyces lactis 304 (±3.15)/ 165.69d* (±0.03)/ 8.53g* (±1.83)/ 496.83a* (±0.10)/ 78.73f* (±2.54)/ 133.68b* (±0.41)/ 292.36a* (±0.07)/ 57.29d* (±0.07)/ 19.83b*
(±0.97) (±0.30) (±0.74) (±0.61) (±0.51) (±0.43) (±0.34) (±0.08)
159.20b 8.34d 458.42d 73.08e 128.91d 302.27b 60.72d 19.12h
Kluyveromyces lactis 317 (±0.60)/ 167.16a* (±0.04)/ 8.76h* (±2.70)/ 481.34b* (±1.34)/ 76.73d* (±0.76)/ 135.55a* (±1.05)/ 317.38a* (±0.35)/ 63.75h* (±0.02)/ 20.08d*
(±0.25) (±0.01) (±1.82) (±0.45) (±0.20) (±0.47) (±0.09) (±0.11)
162.90c 8.25b 494.25b 72.78b 128.67b 300.29b 44.47c 21.38c
Kluyveromyces lactis 318 (±3.25)/ 171.05f* (±0.03)/ 8.67a* (±1.87)/ 518.96d* (±0.27)/ 76.41a* (±0.48)/ 135.11f* (±0.33)/ 315.31a* (±0.88)/ 46.69h* (±0.42)/ 22.45e*
(±1.32) (±0.01) (±3.06) (±0.11) (±1.04) (±0.47) (±0.07) (±0.41)
166.70e 8.57f 498.47a 75.18b 123.19b 299.62f 26.24h 17.96g
Kluyveromyces lactis 325 (±3.06)/ 179.37h* (±0.06)/ 9.19d* (±0.74)/ 533.36b* (±0.28)/ 80.44d* (±0.46)/ 137.16f* (±2.30)/ 320.59a* (±0.03)/ 28.07a* (±0.64)/ 19.21b*
(±0.25) (±0.05) (±2.03) (±0.47) (±1.06) (±0.48) (±0.04) (±0.07)
172.48f 8.03a 202.72g 74.48a 133.29h 232.27e 18.39g 17.56e
Kluyveromyces lactis 469 (±1.33)/ 213.47f* (±0.01)/ 9.39a* (±7.23)/ 237.18b* (±0.11)/ 87.14b* (±0.18)/ 155.94d* (±4.27)/ 271.75h* (±0.65)/ 21.51d* (±0.32)/ 20.54h*
(±1.64) (±0.01) (±0.90) (±0.33) (±0.92) (±0.38) (±0.12) (±0.03)
27.00h 10.61d 85.11h 14.55d 11.84h 29.30h 73.52b 211.11d
Kluyveromyces lactis 868-K (±0.04)/ 27.81a* (±0.06)/ 10.92b* (±0.11)/ 87.66d* (±0.08)/ 14.98h* (±0.01)/ 12.55f* (±0.04)/ 30.18d* (±0.27)/ 75.72a* (±1.24)/ 217.44b*
(±0.04) (±0.04) (±0.51) (±0.03) (±0.10) (±0.18) (±0.11) (±0.83)
170.53b 9.69d 284.42a 29.33g 117.53a 291.60e 64.78a 20.52c
Kluyveromyces lactis 2452 (±0.64)/ 209.75a* (±0.05)/ 11.91b* (±0.42)/ 349.83h* (±1.04)/ 36.07f* (±0.17)/ 144.56b* (±5.36)/ 358.67a* (±0.09)/ 79.68b* (±0.41)/ 25.23d*
(±0.31) (±0.04) (±0.49) (±0.27) (±0.55) (±0.54) (±0.30) (±0.15)
Lsd0.05 0.6815/ 0.2880* 0.0205/ 0.0280 1.0291/ 0.6925 0.2350/ 0.1910* 0.3975/ 0.2980* 1.1575/ 0.3265* 0.1230/ 1.4580* 0.1475/ 0.1125*
SE± 1.363/ 0.576* 0.041/ 0.056* 2.058/ 1.385* 0.470/ 0.382* 0.795/ 0.596* 2.315/ 0.653* 0.246/ 0.161* 0.295/ 0.225*
Notes: a-h Mean ±SD values having the same superscript within a column are insignificantly different (P<0.05). * The data concerning the concentrations of odor-active compounds for control (fermented whey beverages without APF)
The concentrations of odor-active compounds in whey and APF fermented beverages were 3-23 % less than in the control depending on the type of lactose-fermenting yeast strain used as the starter culture. As a result, the fermentation was decreased with the addition of APF. But the influence of APF was the least in the medium based on whey and APF mixture using Kluyveromyces lactis 868-K strain as the starter.
According to previous studies, yeasts can play a major role in the formation of diverse esters during the fermentation process. Ethyl esters, which have pleasant odor notes such as floral, fruity, have generally been reported to occur in fermented beverages. During fermentation, the esterification
of ethanol and organic acids by yeasts can occur, thus conferring fruity and floral odors to fermented beverages [28]. It has been reported that the concentrations of ethyl esters depend on fermentation parameters such as yeast strains, fermentation temperature and concentration of oxygen dissolved in the medium [29]. The contents of methylacetate and ethylacetate were optimal in whey and APF fermented beverage with Kluyveromyces lactis 868-K strain compared with their aroma threshold values for forming of harmonious aroma (10.61 and 85.11 mg/l, respectively). There are low contents of methylacetate in whey and APF fermented beverage with Kluyveromyces lactis 95 and 469 strains (6.69 mg/l and 8.06 mg/l, respectively). Their fruit and floral aroma were
very mild and barely perceptible. The contents of ethylacetate in whey and APF fermented beverage with Kluyveromyces lactis strains coded 42 K, 95, 300, 304, 317, 318, 325, 469 and 2452 were higher in comparison with its aroma threshold values which added sharp flavor of an artificial fruit essence to whey and APF fermented beverages. These high contents indicated that the fermentation was not completed and characterized the beginning of fermentation [30].
Higher alcohols, also known as fusel alcohols, which are formed by fermentation, are important odor-active components in fermented beverages. The amyl alcohols, 3-methyl-1-butanol and 2-methyl-1-butanol, can be formed by the fermentation process from isoleucine and leucine [31]. The increased concentrations of amyl alcohols could be related to the qualities of fermented beverages due to their characteristic fermented, malt-like odor notes. So, the best content of higher alcohols and aldehydes was in whey and APF fermented beverage with Kluyveromyces lactis 868-K which consists of low contents of n-propane (1.84 mg/l), isobutane (29.30 mg/l), acetaldehyde (27.00 mg/l) and high contents of 2-methyl-1-butanol (73.52 mg/l) and 3-methyl-1-butanol (211.11 mg/l). In whey and APF fermented beverage with Kluyveromyces lactis 95 and 469, there are accumulated both the highest contents of n-pro-pane (137.53 mg/l and 133.29 mg/l3, respectively), isobutane (261.80 mg/l and 232.27 mg/l, respectively), acetaldehyde (229.04 mg/l and 172.48 mg/l, respectively), and low contents of 2-methyl-1-butanol (20.78 mg/l and 18.39 mg/l, respectively) and 3-methyl-1-butanol (16.24 mg/l and 17.56 mg/l, respectively) influencing the formation of general aroma. These data described incomplete fermentation too.
7. Conclusions
Based on the study of lactose-fermenting yeasts Kluyveromyces lactis for whey and apple pectin mixture fermentation it was established:
1. The exponential multiplication phase of lactose-fermenting Kluyveromyces lactis strains has shown that maximum biomass accumulation of Kluyveromyces lactis 868-K strain was achieved 71.3*106 CFU/ml compared with other investigated strains.
2. During cultivation in aerobic conditions, maximum biomass accumulation of Kluyveromyces lactis 868-K strain was achieved on the 30 h of cultivation at a temperature of 30±2° С. But the addition of apple pectin in the fiber into whey caused lactose-fermenting yeast growth inhibition (in exponential multiplication phase 1.59 % less biomass accumulation compared with the sample without apple pectin in the fiber). During the alcoholic fermentation, maximum CO2 content and ethanol content of Kluyveromyces lactis 868-K strain were observed after 30 h of fermentation at an optimal temperature of 32° С. The highest ethanol content (0.69 %) was achieved at a temperature of 32 °C with Kluyveromyces lactis 868-K strain. The alcohol content is allowed for soft drinks according to the regulatory requirements.
3. The concentrations of odor-active compounds in whey and APF fermented beverages were 3-23 % less than in the control depending on lactose-fermenting yeast strains. The best contents of higher alcohols, aldehydes and esters were obtained in whey and APF fermented beverage by using Kluyveromyces lactis 868-K strain which consists of low contents of n-propane (1.84 mg/l), isobutane (29.30 mg/l), acetaldehyde (27 mg/l), and high contents of 2-methyl-1-butanol (73.52 mg/l), 3-methyl-1-butanol (211.11 mg/l), methylacetate (10.61 mg/l) and ethylacetate (85.11 mg/l).
Acknowledgments
This work was supported by the National University of Food Technologies, Kyiv, Ukraine (Project № 0112U005376).
References
1. Gonzalez Siso, M. I. The biotechnological utilization of cheese whey: A review [Text] / M. I. Gonzalez Siso // Bioresource Technology. - 1996. - Vol. 57, Issue 1. - P. 1-11. doi: 10.1016/0960-8524(96)00036-3
2. Jelen, P. Whey processing. Utilization and Products [Text] / P. Jelen. - Encyclopedia of Dairy Sciences. 2nd Ed. London: London Academic Press, 2011. - 737 p.
3. Pesta, G. Utilization of whey / Utilization of by-products and treatment of waste in the food industry [Text] / G. Pesta, R. Mey-er-Pittroff, W. Russ. - New York: Springer, 2007. - 1093 p. doi: 10.1007/978-0-387-35766-9
4. Baccouche, A. A physical stability study of whey-based prickly pear beverages [Text] / A. Baccouche, M. Ennouri, I. Felfoul, H. Attia // Food Hydrocolloids. - 2013. - Vol. 33, Issue 2. - P. 234-244. doi: 10.1016/j.foodhyd.2013.03.007
5. Prazeres, A. R. Cheese whey management: A review [Text] / A. R. Prazeres, F. Carvalho, J. Rivas // Journal of Environmental Management. - 2012. - Vol. 110. - P. 48-68. doi: 10.1016/j.jenvman.2012.05.018
6. Hinkova, A. Potential of Membrane Separation Processes in Cheese Whey Fractionation and Separation [Text] / A. Hinkova, P. Zidova, V. Pour, Z. Bubnik, S. Henke, A. Salova, P. Kadlec // Procedia Engineering. - 2012. - Vol. 42. - P. 1425-1436. doi: 10.1016/j.proeng.2012.07.536
7. Pan, K. A study of demineralization of whey by nanofiltration membrane [Text] / K. Pan, Q. Song, L. Wang, B. Cao // Desalination. - 2011. - Vol. 267, Issue 2-3. - P. 217-221. doi: 10.1016/j.desal.2010.09.029
8. Kargi, F. Utilization of cheese whey powder (CWP) for ethanol fermentations: Effects of operating parameters [Text] / F. Kargi, S. Ozmihci // Enzyme and Microbial Technology. - 2006. - Vol. 38, Issue 5. - P. 711-718. doi: 10.1016/j.enzmictec.2005.11.006
9. Rektor, A. Membrane filtration of Mozzarella whey [Text] / A. Rektor, G. Vatai // Desalination. - 2004. - Vol. 162. - P. 279-286. doi: 10.1016/s0011-9164(04)00052-9
10. Jelicic, I. Whey based beverages - new generation of dairy products [Text] / I. Jelicic, R. Bozanic, L. Tratnik // Mljekarstvo. -2008. - Vol. 58. - P. 257-274.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
23.
24.
25.
26.
27.
28.
29.
30.
31.
32.
I
Jiménez-Flores, R. Beverages based on milk fat globule membrane (MFGM) and other novel concepts for dairy- based functional beverages [Text] / R. Jiménez-Flores, I. Higuera-Ciapara, Y. Pouliot // Functional and Speciality Beverage Technology, 2009. -P. 281-296. doi: 10.1533/9781845695569.2.281
Harju, M. Lactose hydrolysis and other conversions in dairy products: Technological aspects [Text] / M. Harju, H. Kallioinen,
0. Tossavainen // International Dairy Journal. - 2012. - Vol. 22, Issue 2. - P. 104-109. doi: 10.1016/j.idairyj.2011.09.011 Dragone, G. (2009). Characterisation of volatile compounds in an alcoholic beverage produced by whey fermentation [Text] / G. Dragone, S. Mussatto, J. Oliveira, J. Teixeira // Food Chemistry. - 2009. - Vol. 112, Issue 4. - P. 929-935. doi: 10.1016/j.food-chem.2008.07.005
Gad, A. S. Utilization Whey in Production of Functional Healthy Beverage "Whey-mango Beverages" [Text] / A. S. Gad, W. H. Emam, G. F. Mohammed, A. F. Sayd // American Journal of Food Technology. - 2013. - Vol. 8, Issue 3. - P. 133-148. doi: 10.3923/ajft.2013.133.148
Pescuma, M. Functional fermented whey-based beverage using lactic acid bacteria [Text] / M. Pescuma, E. M. Hebert, F. Mozzi,
G. F. Valdez // International Journal of Food Microbiology. - 2010. - Vol. 141, Issue 1-2. - P. 73-81. doi: 10.1016/j.ijfoodmi-cro.2010.04.011
Baldasso, C. Concentration and purification of whey proteins by ultrafiltration [Text] / C. Baldasso, T. Barros, I. Tessaro // Desalination. - 2011. - Vol. 278, Issue 1-3. - P. 381-386. doi: 10.1016/j.desal.2011.05.055
Neves, A. R. Overview on sugar metabolism and its control in Lactococcus lactis - The input from in vivo NMR [Text] / A. R. Neves, W. A. Pool, J. Kok, O. P. Kuipers, H. Santos // FEMS Microbiology Reviews. - 2005. - Vol. 29, Issue 3. - P. 531-554. doi: 10.1016/j.fmrre.2005.04.005
Gallardo-Escamilla, F. J. Mouthfeel and flavour of fermented whey with added hydrocolloids [Text] / F. J. Gallardo-Escamilla, A. L. Kelly, C. M. Delahunty // International Dairy Journal. - 2007. - Vol. 17, Issue 4. - P. 308-315. doi: 10.1016/ j.idairyj.2006.04.009
Grek, O. Study of effect fibers on the communication forms of moisture in mixture with milk whey [Text] / O. Grek, O. Krasulya // Maisto chemija ir technologija. - 2013. - Vol. 47. - P. 15-21
Prückler, M. Wheat bran-based biorefinery 1: Composition of wheat bran and strategies of functionalization [Text] / M. Prückler, S. Siebenhandlehn, S. Apprich, S. Holtinger, C. Haas, E. Schmid, W. Kneifel // LWT - Food Science and Technology. - 2014. -Vol. 56, Issue 2. - P. 211-221. doi: 10.1016/j.lwt.2013.12.004
Kohajdova, Z. Effect of apple pomace powder addition on farinographic properties of wheat dough and biscuits quality [Text] / Z. Kohajdova, J. Karovicova, M. Magala, V. Kuchtova // Chemical Papers. - 2014. - Vol. 68, Issue 8. - P. 1059-1065. doi: 10.2478/ s11696-014-0567-1
Min, B. Utilization of pectin-enriched materials from apple pomace as a fat replacer in a model food system [Text] / B. Min,
1. Y. Bae, H. G. Lee, S. H. Yoo, S. Lee // Bioresource Technology. - 2010. - Vol. 101, Issue 14. - P. 5414-5418. doi: 10.1016/ j.biortech.2010.02.022
Matijevic, B. Impact of enzymatic hydrolyzed lactose on fermentation and growth of probiotic bacteria in whey [Text] / B. Matije-vic, K. Lisak, R. Bozanic, L. Tratnik // Mljekarstvo. - 2011. - Vol. 61, Issue 2. - P. 154-160.
Lima, A. F. Comparative biochemical characterization of soluble and chitosan immobilized p-galactosidase from Kluyveromyces-lactis NRRL Y1564 [Text] / A. F. Lima, K. F. Cavalcante, M. D. Freitas, T. H. Rodrigues, M. V. Rocha, L. R. Goncalves // Process Biochemistry. - 2013. - Vol. 48, Issue 3. - P. 443-452. doi: 10.1016/j.procbio.2013.02.002
Ukraine State Standard 4069:2002. Soft drinks. General specifications [Text]. - Derzhspozhyvstandard of Ukraine Publishing, Kyiv, 2002. - Available at: http://dobavkam.net
Park, M. K. Study of volatile organic acids in freeze-dried Cheonggukjang formed during fermentation using SPME and stable-isotope dilution assay (SIDA) [Text] / M. K. Park, H.-K. Choi, D.-Y. Kwon, Y.-S. Kim // Food Chemistry. - 2007. - Vol. 105, Issue 3. - P. 1276-1280. doi: 10.1016/j.foodchem.2007.03.012
Azhu Valappil, Z. Impact of thermal and nonthermal processing technologies on unfermented apple cider aroma volatiles [Text] / Z. Azhu Valappil, X. Fan, H. Q. Zhang, R. L. Rouseff // Journal of Agricultural and Food Chemistry. - 2009. - Vol. 57, Issue 3. -P. 924-929. doi: 10.1021/jf803142d
Plutowska, B. Application of gas chromatography-olfactometry (GC-O) in analysis and quality assessment of alcoholic beverages - A review [Text] / B. Plutowska, W. Wardencki // Food Chemistry. - 2008. - Vol. 107, Issue 1. - P. 449-463. doi: 10.1016/ j.foodchem.2007.08.058
Vanderhaegen, B. Evolution of Chemical and Sensory Properties during Aging of Top-Fermented Beer [Text] / B. Vanderhaegen,
H. Neven, S. Coghe, K. J. Verstrepen, H. Verachtert, G. Derdelinckx // Journal of Agricultural and Food Chemistry. - 2003. -Vol. 51, Issue 23. - P. 6782-6790. doi: 10.1021/jf034631z
Duarte, W. F. Raspberry (Rubus idaeus L.) wine: Yeast selection, sensory evaluation and instrumental analysis of volatile and other compounds [Text] / W. F. Duarte, D. R. Dias, J. M. Oliveira, M. Vilanova, J. A. Teixeira, J. B. A. Silva, R. F. Schwan // Food Research International. - 2010. - Vol. 43, Issue 9. - P. 2303-2314. doi: 10.1016/j.foodres.2010.08.003
Marinchenko, V. O. Technology of ethanol [Text] / V. O. Marinchenko, V. A. Domaretskiy, P. L. Shiyan. - Vinnitsa: Podillay-2000, 2003. - 496 p.
Pino, J. A. Analysis of volatile compounds of mango wine [Text] / J. A. Pino, O. Queris // Food Chemistry. - 2011. - Vol. 125, Issue 4. - P. 1141-1146. doi: 10.1016/j.foodchem.2010.09.056
