On the influence of non-enzymatic crosslinking of caseins on the gel strength of yoghurt.

After storage of UHT milk at 37 degrees C resp. 50 degrees C, yoghurt was prepared. For a storage temperature of 37 degrees C, breaking strength of the yoghurt samples increased from 2.7 to 5.8 N with increasing storage duration of the UHT milk. A plateau is reached after 17 days of storage. This increase in breaking strength correlates with a significant increase in non-reducible casein oligomerization from 14% for fresh UHT milk to 25% measured using size exclusion chromatography under reducing and denaturing conditions and calculated as sum of predominantly formed dimers and trimers at the total casein fraction. At a storage temperature of 50 degrees C, a less increase in breaking strength from 2.7 to 4.6 N with a plateau after 17 days was observed while casein oligomerization increased to 63%. After acid hydrolysis, only lysinoalanine and histidinoalanine were detected in the caseinate samples via amino acid analysis. The quantified concentration of lysinoalanine and histidinoalanine could not explain the observed casein oligomerization. Thus, unknown crosslinked amino acids must have been formed during storage, inducing significant changes in the functional properties of milk proteins.     

Extent of damage to amino acid availability of whey protein heated with sugar.

The effect of heat treatments, at various water activities (aW), on digestibility and on the availabilities of amino acids of whey protein samples in the presence of lactose was estimated by an in vitro digestion method with continuous dialysis. Four aW (0.3, 0.5, 0.7 and 0.97), three temperatures (75, 100 and 121 degrees C) and three heating periods (50, 500 and 5000 s) were selected. The initial lysine:lactose molar ratio was 1:1. Amino acid profiles showed that excessive heating of whey (121 degrees C, 5000 s) destroyed a significant proportion of cystine at all aW, lysine at aW 0.3, 0.5 and 0.7, and arginine at aW 0.5 and 0.7. At aW 0.3, 0.5 and 0.7, protein digestibility decreased (P less than 0.05) as the temperature increased from 75 to 121 degrees C for a heating period of 5000 s, and as the heating time was prolonged, from 500 to 5000 s at 121 degrees C. Excessive heating also decreased (P less than 0.05) the availabilities of all amino acids at aW 0.3, 0.5 and 0.7. The availabilities of lysine, proline, aspartic acid, glutamic acid, threonine, alanine, glycine and serine were particularly affected. Severe heating at aW 0.97 did not seem to favour the Maillard reaction, but the availabilities of cystine, tyrosine and arginine were decreased, probably as a result of structural modifications of the protein upon heating. Heating whey protein concentrates in the presence of lactose not only affected lysine, but also impaired enzymic liberation of other amino acids, according to the severity of heat treatments and aW.     

Selective fortification of lysinoalanine, fructoselysine and N epsilon-carboxymethyllysine in casein model systems.

In the present study, a promising strategy to study nutritional effects of selected chemical reaction products formed in heat treated protein containing foods is addressed. In due course, a selective fortification of different marker compounds for lysine damage in casein-sugar mixtures was performed to provide model systems being applicable to investigate biological effects of the cross-link lysinoalanine (LAL), the MRPs fructoselysine (FL) and N epsilon-carboxymethyllysine (CML) in a casein-linked preparation. The three different model proteins, casein-LAL, casein-FL and casein-CML were prepared by heating casein either in strong alkaline conditions at 105 degrees C for 1 h, in the presence of glucose at 65 degrees C for 68 h, or in the presence of glyoxylic acid at 37 degrees C for 19 h. Finally, the degree of lysine modification achieved was 39%, 75% and 55% for the casein-LAL, casein-FL and casein-CML, respectively. The calculation of lysine recovery and the respective analysis of each single modified casein (LAL-, FL- and CML-MP) for the selected fortified compound and each other compound vice versa proved that the individual procedure provides a specific fortification for LAL, FL and CML, respectively. The modified proteins are suitable as reference model proteins to be investigated for specific biological and toxicological effects of casein-linked LAL, FL and CML.     

Formation of dehydroalanine, lanthionine and lysinoalanine during heat treatment of beta-lactoglobuline A (author's transl)]

Solutions of beta-lactoglobulin A were heated at 97.5 degrees C at pH 6.9 and 9.8 80 min. The formation of dehydroalanine, lanthionine and lysinoalanine as well as changes in the levels of cysteine, cystine and the basic amino acids were recorded. Lanthionine (equal amounts of the 1- and the mesoform) was formed only in small amounts, while the lysinoalanine levels were rather high. Beside this, lysine was converted to unknown compounds. The main sources for the formation of dehydroalanine are the cystine moieties of the protein. Cysteine residues are first oxidized to cystine before beta-elimination occurs.     

Effect of Heat Treatments on In Vitro Digestibility of Delactosed Whey Protein as Determined by the Digestion Cell Technique

Diafiltered whey protein concentrates (WPC) were heated, in liquid form, under various conditions of time and temperature. Protein and amino acid digestibilities were determined by in vitro pepsin and pan-creatin digestion with continuous dialysis. Whey nitrogen digestibility decreased (P < 0.05) after heating at 121°C for 5000 sec. Heating WPC for 5000 set at temperatures below 121°C increased the digestibility of all amino acids except alanine and lysine at 75°C, and methionine and lysine at 100°C. In general, the longer was the heating period at 100°C or 121°C the lower was the amino acid digestibility. These results emphasized the importance of structural modifications brought about by heat treatments on protein digestibility.     

On the influence of non‐enzymatic crosslinking of caseins on the gel strength of yoghurt

After storage of UHT milk at 37°C resp. 50°C, yoghurt was prepared. For a storage temperature of 37°C, breaking strength of the yoghurt samples increased from 2.7 to 5.8 N with increasing storage duration of the UHT milk. A plateau is reached after 17 days of storage. This increase in breaking strength correlates with a significant increase in non‐reducible casein oligomerization from 14% for fresh UHT milk to 25% measured using size exclusion chromatography under reducing and denaturing conditions and calculated as sum of predominantly formed dimers and trimers at the total casein fraction. At a storage temperature of 50°C, a less increase in breaking strength from 2.7 to 4.6 N with a plateau after 17 days was observed while casein oligomerization increased to 63%. After acid hydrolysis, only lysinoalanine and histidinoalanine were detected in the caseinate samples via amino acid analysis. The quantified concentration of lysinoalanine and histidinoalanine could not explain the observed casein oligomerization. Thus, unknown crosslinked amino acids must have been formed during storage, inducing significant changes in the functional properties of milk proteins.     

A new approach to microwave food research: Analyzing the electromagnetic response of basic amino acids

Measurement of the electromagnetic properties of food is important for its electromagnetic protection, microwave heating and sterilization. Much research had focused on describing the non-thermal mechanism of electromagnetism and locating its site of action. Amino acids are the basic units of proteins, and therefore their electromagnetic characteristics are central to understanding the transmission and loss of microwave energy in food and the non-thermal mechanism of electromagnetic effects. Herein, the dielectric properties and conductivity characteristics of solutions of basic amino acids, at an electromagnetic frequency of 2.45 GHz and at temperatures between 10 °C and 70 °C, have been measured. The results show that the dielectric constant and dielectric loss decrease with increasing temperature, for example, increase the temperature form 10 °C to 70 °C, the dielectric constant of lysine at 2% decreased from 77.259 to 68.601, and the dielectric loss decreased from 13.412 to 10.302, due to the decrease of the solution viscosity and the relaxation time, causing an increase of the dipole moment. Increasing the concentration of the basic amino acids leads to an increase of the dielectric loss, fix the temperature at 20 °C, the concentration of lysine increased from 0.5% to 10%, the dielectric loss increased from 10.191 to 23.232, which is related to the change of the conductivity. The conductivity has positive correlation with the concentration and temperature of the amino acid solution, fix the temperature at 20 °C, the concentration of lysine increased from 0.5% to 10%, the conductivity increased from 234 to 2840 μS/cm, and when the temperature was increased form 10 °C to 70 °C, the conductivity of lysine at 2% increased from 694 to 1347 μS/cm. The absorption properties of the solutions are characterized by the loss of reflection, and the absorption characteristics of the basic amino acids are related to the electromagnetic frequency, the concentration and the thickness of the absorbing layer, the variation of which affects the impedance matching. In this paper, through the study of the electromagnetic characteristics of basic amino acids in solution, a theoretical basis for the application of basic amino acids in microwave sterilization, as well as a mechanism for the microwave effect, are proposed.     

Changes in the amino acid compounds of the wholecrop wheat during ensiling and after fermentation

Changes in amino acid fractions of chopped low-to-medium protein whole-crop wheat were studied at four stages of maturation and at two stages after a 24 h wilting period. Tests were carried out after an ensiling period of 90 days, and after a further 7-day post-fermentation aerobic exposure. Total amino acids contents in the dry matter during the fermentation period remained stable. Quantities of essential amino acids decreased during maturation and fermentation and remained stable during post fermentation aerobic exposure. The highest amino acid concentrations recorded in the fresh material were those of arginine, lysine, glutamic acid, alanine, leucine, glycine and aspartic acid. Free amino acids varied from a low level in the fresh material < 10% of the total amino acids to ca 65% after the aerobic process. Ammonia-N content increased in the silage and remained relatively stable on post fermentation aerobic exposure. The concentration of all free amino acids increased except arginine and glutamine during the ensiling period and post fermentation aerobic exposure. After wilting, an increase in free amino acids and a decrease in insoluble amino acids was recorded. The changes in concentration of 18 amino acids during ensilage and following post fermentation aerobic exposure are given.     

Nε-carboxymethyllysine is formed during heating of lysine with ketoses

In a model system with lysine monohydrochloride and various reducing sugars, the contents of N^ε-carboxymethyllysine (CML) were measured after heat treatment at 98°C for 3–24 h. The results show for the first time that CML could be formed by the reaction of lysine and fructose or lysine and sorbose. The formation of CML also shows time-dependent differences between aldoses and ketoses. After heating up to 6–12 h, the reaction of lysine with ketoses leads to similar amounts of CML as the reaction with aldoses, whereas after longer heating significantly more CML was produced by the reaction with aldoses. The highest CML contents (2380 mg CML kg^?1 lysine) were measured in the galactose model system after heating for 24 h.     

INSTABILITY OF SODIUM NITRITE IN A CHEMICALLY DEFINED MICROBIOLOGICAL MEDIUM

Mixtures of sodium nitrite, amino acids and ascorbic acid at pH 6.3 or 7.2 were filter sterilized or heated for various times at 121°C. Samples were analyzed for nitrite concentration immediately after treatment and after storage at 37 or 43°C. Heating the complete medium for 15 min at 121°C reduced the nitrite concentration by 30–50% independent of the initial nitrite concentration. Storage of complete filter sterilized medium at 43°C for 18–24 hr resulted in 50–65% loss of nitrite while in heat sterilized medium the loss was as great as 90%. None of the 19 amino acids stored individually with sodium nitrite at 37°C for up to 4 hr affected the nitrite concentration, whereas ascorbic acid resulted in total disappearance of nitrite. During heating at 121°C of the 19 amino acids only cystine resulted in any significant nitrite loss after 15 min of treatment while ascorbic acid effected total nitrite disappearance after 5 min. The defined medium containing nitrite demonstrated inhibitory activity against seven strains of Clostridium perfringens.     

Lethality of commercial whole-muscle beef jerky manufacturing processes against Salmonella serovars and Escherichia coli O157:H7

Thermal processes used in making whole-muscle beef jerky include a drying step, which may result in enhanced pathogen thermotolerance and evaporative cooling that reduce process lethality. Several salmonellosis outbreaks have been associated with beef jerky. In this study, a standardized process was used to inoculate beef strips with five-strain cocktails of either Salmonella serovars or Escherichia coli O157:H7, to marinate the strips at pH 5.3 for 22 to 24 h at 5 degrees C, and to convert the strips to jerky using various heating and drying regimes. Numbers of surviving organisms were determined during and after heating and drying. Salmonella reductions of > or = 6.4 log CFU and similar reductions in E. coli O157:H7 were best achieved by ensuring that high wet-bulb temperatures were reached and maintained early in the process (51.7 or 54.4 degrees C for 60 min, 57.2 degrees C for 30 min, or 60 degrees C for 10 min) followed by drying at 76.7 degrees C (dry-bulb temperature). Processes with less lethality that reduced counts of both pathogens by > or = 5.0 log CFU were (i) heating and drying at 76.7 degrees C (dry bulb) within 90 min of beginning the process, (ii) heating for successive hourly intervals at 48.9, 54.4, 60, and 76.7 degrees C (dry bulb), and (iii) heating at 51.7 degrees C (dry bulb) and then drying at 76.7 degrees C (dry bulb), starting before the product water activity dropped below 0.86. In several trials, separate beef strips were inoculated with a commercial Pediococcus acidilactici starter culture as a potential surrogate for evaluating pathogen thermotolerance. The results of these trials suggested that this experimental approach may be useful for in-plant validation of process lethality.     

Study of nonenzymic browning in α-amino acid and γ-aminobutyric acid/sugar model systems

The reactivities, in nonenzymic browning, of γ-aminobutyric acid (GABA), a non-protein amino acid with wide natural occurrence and potential health benefits as it occurs in foods, and of the α-l-amino acids arginine, glutamic acid, glutamine, leucine, lysine, and phenylalanine were investigated by heating equimolar mixtures of glucose and the cited amino acids at 110 °C at pH 6.0 for different times (0–4 h). Linear regression analysis indicated that the colour development in a GABA/glucose mixture was slower than that of a lysine/glucose mixture and comparable to that of a phenylalanine/glucose mixture. High-performance anion-exchange chromatography (HPAEC) with integrated pulsed amperometric detection (IPAD) showed that the decrease in GABA levels (ca. 10% after heating for 4 h) as a function of heating time was smaller than that of glucose (ca. 30% after heating for 4 h). At the same time, glucose to fructose isomerisation took place. After 20 min of heating at pH 6.0, all mixtures showed a fructose peak, the area of which increased with heating time. However, after correcting for fructose isomerisation, glucose losses were still higher than amino acid losses. In contrast to its precursor glutamic acid, GABA was stable during heating of a solution containing it alone. Heating of GABA-containing d-sugar solutions (xylose, fructose, glucose, maltose and sucrose) showed that the relative order of colour yield was pentose > hexose > disaccharides. As well as glucose to fructose isomerisation, HPAEC–IPAD allowed monitoring of the different isomerisation reactions occurring, and also disaccharide hydrolysis in the different GABA/sugar mixtures.     

通电加热过程中凡纳滨对虾虾肉糜的游离氨基酸和核苷酸含量变化研究

为研究通电加热过程中不同加热温度(40、55、65、75、85和95℃)对凡纳滨对虾虾肉糜中主要非挥发性滋味成分的影响,采用氨基酸自动分析仪和高效液相色谱仪分别检测了虾肉糜中的游离氨基酸和核苷酸及其关联产物含量,并采用味道强度值(TAV)确定其中主要的呈味物质及贡献程度,最后通过味精当量(EUC)分析鲜味氨基酸和呈味核苷酸之间的协同作用,并对其鲜味进行评价。结果表明,与鲜虾相比,通电热处理的虾肉糜中游离氨基酸总量均有不同程度的减少。随着温度的升高,鲜味氨基酸、甜味氨基酸和苦味氨基酸的含量均呈现先减少后增加的趋势,且在65℃达到最小值(鲜味氨基酸、甜味氨基酸和苦味氨基酸的总TAV值分别为0.86、4.40、4.46)。通电加热并不影响ATP的降解途径只是会改变核苷酸及其关联产物的相对含量,尤其是促进Ado的产生。甘氨酸、精氨酸、谷氨酸、AMP和IMP在不同加热温度下其TAV几乎都大于1,是虾肉糜滋味的主要贡献者。味精当量以谷氨酸钠质量计,40℃最能促进虾肉糜鲜味的产生(EUC值为5.54 g MSG/100 g),在85℃处虾肉糜风味损失最小(EUC值为4.02 g MSG/100 g)。     

Reduction of radiation-induced vitamin E- and B1- losses by irradiation of foodstuffs at low temperature and by exclusion of atmospheric oxygen

The protective effect of low temperatures during irradiation on vitamin B1 and E levels in foods is not abolished by subsequent storage or heating. Egg powder irradiated at 1 Mrad in the presence of air and stored for 4 months at ambient temperature lost 68% of its thiamin content when irradiated at 20 degrees C, 33% when irradiated at -30 degrees C. Sunflower oil irradiated at 3 Mrad in the presence of air and subsequently heated for 1 hour at 180 degrees C lost 98% of its alpha-tocopherol content when irradiated at 20 degrees C, 65% when irradiated at -30 degrees C. Exclusion of atmospheric oxygen by packaging under nitrogen reduced the loss of alpha-tocopherol in irradiated (0.1 Mrad) rolled oats after 8 months of storage from 56 to 5% and the loss of thiamin from 86 to 26%. Vacuum packaging was equally effective during the first 3 months and somewhat less effective during the following 5 months. Packaging under carbon dioxide showed no advantage over packaging in air. Sensory evaluation of rolled oats, raw or cooked, 1 and 3 months after irradiation with 0.1 Mrad indicated no significant quality difference between unirradiated and irradiated samples packaged under nitrogen.     

Effects of alkali treatment on the in-vitro digestibility of proteins and the release of amino acids

Three protein sources, casein, soya bean and rapeseed concentrates, were subjected to alkali treatment (0.2 M, 60° C) for 2 or 6 h. The impact of these treatments on protein digestibility and on release of amino acids, especially lysinoalanine, was evaluated by an in-vitro enzymic digestion method with simultaneous dialysis of digestion products. The impairment of digestibility was higher for casein and soya bean concentrate than for rapeseed concentrate. Whatever the amount of lysinoalanine produced in each protein, it was poorly released by proteolytic enzymes. The rate of release of other amino acids was reduced by the treatments, but to different levels for each protein. Arginine and lysine were particularly affected. As can be inferred from the release of the target amino acids, the hydrolytic capacity of chymotrypsin was not specifically impaired, in contrast to that of trypsin for casein and of elastase for all protein sources. This technique was useful to evaluate quickly the effects of processing on the digestibility of proteins.     

高效液相色谱法检测食品中17种游离氨基酸

目的使用异硫氰酸苯酯衍生-高效液相色谱法对食品中17种游离氨基酸进行检测。方法对17种游离氨基酸及其苯胺硫甲酰基衍生物稳定性的分析,研究游离氨基酸的最佳衍生条件。同时,对盐沉淀法和C_(18)固相萃取(solid phase extraction, SPE)小柱法2种样品除杂方法的加标回收率进行测定。结果胱氨酸加热干燥5h后,回收率为68.46%±4.01%,热稳定性差;其它16种氨基酸加热1～5h后回收率均在90%～110%之间,热稳定性较好。胱氨酸的苯胺硫甲酰基衍生物,6h后回收率仅为11.04%±1.65%,稳定性差;其它氨基酸的苯胺硫甲酰基衍生物24 h后回收率均大于90%,比较稳定。另外,前处理中使用C_(18) SPE除杂时,样品中游离氨基酸的加标回收率均大于70%,而使用水杨酸和三氯乙酸除蛋白后苯胺硫甲酰基衍生物降解严重。结论除胱氨酸外,其余16种氨基酸的热稳定性均较好,且生成的苯胺硫甲酰基衍生物比较稳定,可以通过异硫氰酸苯酯衍生进行定量分析。相比于水杨酸和三氯乙酸沉淀法, C_(18) SPE柱是游离氨基酸除杂方法的首选,而无机盐影响异硫氰酸苯酯与氨基酸衍生,因此此方法不适用于含盐样品的衍生。     

The contents of lignans in commercial sesame oils of Taiwan and their changes during heating

Sesame lignans have multiple functions and were recently reported to have potential as sources of phytoestrogens. Sesame oils used in Taiwan are expelled from roasted sesame seeds with dark colour and strong flavour. This study analyzed lignan contents of 14 brands of sesame oils, and found their mean of total lignans to be 11.5 mg/g; 82% and 15% of the lignans were sesamin, and sesamolin, respectively. Sesamol contents were relatively higher in those with darker colour. In use as a cooking oil, heating at 180 °C for 4 min did not change the content of lignans, but the level of sesamol increased after heating at 180 °C for 20 min. Heating at 200 °C for 20 min caused a significant loss of sesamolin and sesamol. From our calculation, ingestion of 10 g of sesame oil is adequate to provide the level of lignans that might benefit cardiovascular health, as found by other studies. Cooking at temperatures above 200 °C will cause loss of some lignans, but sesamin, a source of phytoestrogen, is relatively heat-stable.     

Nonprotein Nitrogen (NPN) and Free amino acid contents of dry, fermented and nonfermented sausages

Fermented and nonfermented sausages were analyzed for concentration changes in total and nonprotein nitrogen (NPN) and in free amino acid profiles during processing. Sausages fermented by Pediococcus pentaceous at 38°C and heated to 60°C had increases (p < 0·05) in total N and NPN contents and increases in concentration (≥5 mg per 100 g dry sample) for 14 of 20 free amino acids. Nonfermented sausages had no increase (p > 0·05) of NPN content and increases in only 6 of 20 free amino acids after heating to 60°C. When the effects of three starter cultures were compared, NPN concentrations after fermentation and heating and after drying were highest to lowest in the following order: P. pentaceous >P. acidilactici with M. varians >P. acidilactici. Variations of free amino acid patterns in the fermented, dried sausages were found which indicated differences in metabolic activity between the cultures. These variations did not (p > 0·05) affect sensory scores for the dried sausages. Overall, these results for rapidly fermented and mildly heated dry sausages were similar to results previously reported for 'European-style' dry sausages processed with prolonged low temperature fermentations.     

Effect of dry heating on the microbiological quality,functional properties,and natural bacteriostatic ability of egg white after reconstitution

Spray-dried egg white (powder) is widely used in the food industry because of its variety of functional properties and its practical advantages. Moreover, egg white powder is generally considered safe because it can withstand high temperatures that allow for the destruction of all pathogens, especially Salmonella. In France, two types of treatments are used to improve the functional properties (whipping and gelling) of dried egg white: standard storage at 67 degrees C for about 15 days and storage at 75 to 80 degrees C for 15 days. The objective of this study was to investigate the effects of two dry-heating treatments (storage at 67 and 75 degrees C for 15 days) on the subsequent ability of egg white to resist Salmonella growth after reconstitution. The impact on the endogenous microflora of the powder and on its functional properties was also considered. Both dry-heating treatments were efficient in destroying a large number of Salmonella. Dry heating at 75 degrees C affected the bacteriostatic ability of reconstituted egg white to a greater extent than did dry heating at 67 degrees C. This loss of bacteriostatic ability could be attributable to the thermal denaturation of ovotransferrin, resulting in a reduction in its activity as an iron chelator. However, dry heating at 75 degrees C resulted in improved functional properties. Ultimately, no complete compromise between better functional quality and the preservation of the bacteriostatic ability of egg white after reconstitution is possible. Our results underline the importance of the use of hygienic conditions with egg white powder, especially with powder subjected to high-temperature treatments.     

Amino acid profiles of lactic acid bacteria, isolated from kefir grains and kefir starter made from them

The characteristics of cell growth, lactic acid production, amino acid release and consumption by single-strain cultures of lactic acid bacteria (isolated from kefir grains), and by a multiple-strain kefir starter prepared from them, were studied. The change in the levels of free amino acids was followed throughout the kefir process: single-strain kefir bacteria and the kefir starter (Lactococcus lactis C15-1%+Lactobacillus helveticus MP12-3%+(Streptococcus thermophilus T15+Lactobacillus bulgaricus HP1 = 1:1)-3%) were cultivated in pasteurized (92 degrees C for 20 min) cow's milk (3% fat content) at 28 degrees C for 5 h (the kefir starter reached pH 4.7) and subsequently grown at 20 degrees C for 16 h; storage was at 4 degrees C for 168 h. The strain L. helveticus MP12 was unrivaled with respect to free amino acid production (53.38 mg (100 g)(-1)) and cell growth (17.8 x 10(8) CFU ml(-1)); however, it manifested the lowest acidification activity. L. bulgaricus HP1 released approximately 3.7 times less amino acids, nearly 5 times lower cell growth, and produced about 1.2 times more lactic acid. S. thermophilus T15 demonstrated dramatically complex amino acid necessities for growth and metabolism. With L. lactis C15, the highest levels of growth and lactic acid synthesis were recorded (18.3 x 10(8) CFU ml(-1) and 7.8 g l(-1) lactic acid at the 21st hour), and as for free amino acid production, it approximated L. bulgaricus HP1 (17.03 mg (100 g)(-1) maximum concentration). In the L. lactis C15 culture, the amino acids were used more actively throughout the first exponential growth phase (by the 10th hour) than during the second growth phase. The unique properties of the L. helveticus MP12 strain to produce amino acids were employed to create a symbiotic bioconsortium kefir culture, which, under conditions of kefir formation, enhanced lactic acid production and shortened the time required to reach pH 4.7; intensified cell growth activity, resulting in a respective 90- and 60-fold increase in the concentration of lactobacilli and cocci in the mixed culture compared to individual cultures; and accumulated free amino acids in the final kefir with higher total concentrations (56.88 mg (100 g)(-1)) and an individual concentration of essential amino acids (1.5 times) greater than that of yogurt.