3.1. The Growth and Acidification Performance of LAB
As shown in Figure 1
, the total LAB count in GY and GYY both increased during fermentation and exceeded the minimum bacteria populations (106
cfu/mL), which are required for probiotic foods to possess health claims [24
]. It was noticed that the population of total LAB was significantly (p
< 0.05) higher in multi-starters fermentation system (log 9.65 CFU/mL) than single culture (log 9.45 CFU/mL) at the end of 4 h fermentation. This was in consistent with Chaves-López et al. [25
], who reported that co-culturing with yeasts was able to promote the multiplication of LAB. This phenomenon might be attributed to the release of amino acids and vitamins through the metabolism of yeast [26
], which might provide more nutrients for the growth of LAB.
Acidity is an important quality indicator of fermented milk, which is closely related to the texture and flavor of the product [27
]. Appropriate acidity gives the product a unique flavor and inhibits the growth of spoilage bacteria and food-borne pathogens [28
]. The curves of pH and titratable acidity changes are presented in Figure 2
. The pH values decreased while titratable acidity increased during the period of fermentation for both GY and GYY, mostly due to the accumulation of lactic acid by the metabolism of LAB. By contrast, GYY presented lower pH values but higher titratable acidity values than GY during fermentation process, and the gap of pH and titratable acidity between GY and GYY increased as a function of fermentation time. Lower titratable acidity and higher pH value were observed in co-cultures with yeasts and LAB than single culture of LAB. Similar observations were found in skim milk co-cultured with K. marxianus
and L. helveticus
for 12 h, resulted in a significantly lower pH range (6.43 to 4.00) compared to pH 4.19 when L. helveticus
was grown alone [29
]. In conjunction with the results in Figure 1
and Figure 2
, addition of K. marxianus
probably stimulates the growth of LAB to increase their acid production.
3.2. Rheological Properties of GM, GY, and GYY
As shown in Figure 3
a, the apparent viscosity of goat milk and yogurts decreased with the increase of shear rate. All milk and yogurt samples exhibited pseudoplastic behavior, and fermentation could enhance the apparent viscosity of goat milk. GYY had the highest viscosity compared with GM and GY throughout the whole range of shear rate. LAB produce lactate as they grow, and lactate accumulation leads to a decrease in goat milk pH. This could promote the association of casein micelles, leading to the increase of the apparent viscosity of goat milk during fermentation. Exopolysaccharide produced by LAB was reported to improve texture and sensory characteristics of yogurt, such as shininess, creaminess, ropiness, and mouthful [30
]. Co-fermented goat milk with LAB and yeast significantly (p
< 0.05) improved the apparent viscosity compared with fermented goat milk with LAB, might due to the stimulation of the growth of LAB by K. marxianus
and more exopolysaccharide produced in GYY. Two rheological factors elastic modulus (G′) and viscous modulus (G″) were observed during frequency sweep test, which could reflect the elastic and viscous properties of the gels. As shown in Figure 3
b, GM, GY, and GYY were both increased in a frequency-dependent manner. GY and GYY exhibited typical characteristics of weak viscoelastic gels as G′ were both higher than their corresponding G″ over all frequency range. In addition, GYY had the highest G′ and G″ whereas GM exhibited the lowest G′ and G″, indicating that GYY had the highest degree of elastic and viscous character. The increase of Gʹ and G″ during fermentation could be attributed to the metabolic activity of LAB, which accelerated the association of casein micelles by decreasing the pH of goat milk [31
]. This phenomenon might also be explained by a large amount of exopolysaccharide produced by LAB in GYY. A similar observation was reported by Bensmira et al. [32
] who attributed the increase of G′ and G″ in kefir to the accumulation of exopolysaccharide generated by LAB.
3.4. Volatile Organic Compounds in GM, GY, and GYY from SPME-GC-MS
Significant differences (p
< 0.05) of aroma compounds were found among GM, GY, and GYY on the basis of E-nose data. Hence, GC-MS was applied to further confirm the specific VOC in samples. A total six groups of volatiles were identified in GM, GY, and GYY by GC-MS; VOC contents were expressed as log10 [peak area of respective volatile organic compound in arbitrary unit] [35
], and relative content (RC) was summarized in Table 2
In order to obtain the interrelationships among six groups of volatiles (acids, alcohols, esters, aldehydes, ketone, and phenols) and samples, PCA was applied and result was shown in Figure 6
. The two principal components described most of the original variance in the data set according to the accumulated contribution of the first two principal components, which was 99.99%. The PC1 represented the main part of the variance (92%) while PC2 represented 7.99% of the original variance. Obviously, PC1 correlated well with acids, alcohols, esters, ketones, and GYY, which indicated that more acids, alcohols, esters, and ketones were detected in GYY. The content of acids, alcohols, esters and ketones were found to correlate well according to their similar loadings on PC1. Conversely, the negative correlation between PC1 and GM suggested that the low content of acids, alcohols, esters and ketone in GM. Similarly, GM had higher content of phenols and aldehydes than GYY due to the good correlation with phenols and aldehydes as shown in Figure 6
. According to the PCA, high ketones, acids, alcohols and esters were generated during fermentation.
As shown in Table 2
, caproic, caprylic, and capric acid were both detected in GM, GY, and GYY. For GM and GYY, caproic and caprylic increased from 8.56 ± 0.03 to 9.16 ± 0.04 and from 8.54 ± 0.05 to 9.26 ± 0.09, respectively, during fermentation. Capric acid decreased to 8.58 ± 0.07 in GY whereas increased to 9.35 ± 0.08 in GYY. The increase of free fatty acid content during fermentation probably attributed to the breakdown of amino acids and fat lipolysis in goat milk by the metabolism of microflora [36
]. However, the relative content of caproic, caprylic, and capric acid decreased in the process of fermentation. As shown in Table 2
, comparing GM with GYY, the relative content of caproic, caprylic, and capric acid decreased from 7.94% to 5.68%, from 7.91% to 5.74% and from 8.03% to 5.80%, respectively. Acetic acid was reported as a factor which contributed to the pungent smell in kefir fermented goat milk [37
]. In this study, acetic acid was found both in GM and GY whereas decreased to no detected levels in GYY, mainly due to the reaction of acetic acid with ethyl alcohol to form ethyl acetate. Thus, plenty of ethyl acetate was detected in GYY, and ethyl acetate was known for its formation of a fruity character. Isoamyl acetate (RPA = 5.22%) with the banana flavor was identified in GYY, which probably formed by the reaction of acetic acid and 2-methyl-2,4-pentanediol. The relative content of total esters was 16.15% in GYY while the vast majority of esters are described as possessing fruity, sweet and floral note. Hence, esters were considered as the key VOC which contributed to the pleasant flavor of GYY.
Ethyl alcohol was commonly detected in the yeast-lactic fermented milk (such as kefir), though there was no ethanol detected in this study. Ethanol produced by K. marxianus
in the process of alcoholic fermentation might all transfer into esters and other substances, or perhaps no ethanol is accumulated because of the short fermentation time. As shown in Figure 6
and Table 2
, we could clearly find out that the relative peak area of phenethyl alcohol, isooctyl alcohol and undecyl alcohol increased to 5.75%, 4.99%, and 5.11%, respectively. Phenethyl alcohol is generally believed to possess the scent of honey and rose and play a crucial role in beer as an aromatic alcohol compound.
Methylnonylketone (RPA = 5.05%) was also identified in GYY as a characteristic aroma, which possess a pleasant smell and generally found in goat chess and fermented fruit juice [38
]. The relative contents of aldehydes in GM, such as hexanal (green grass aroma), nonanal (slightly rancid), benzenepropanal (balsam aroma) and undecanal (rose aroma), were 7.65%, 7.94%, 7.92%, and 6.88%, respectively. All the aldehydes in goat milk might be converted to acids or alcohols through fermentation, which resulted in no detected aldehydes in GYY.
3.5. Sensory Properties of Two Different Goat Yogurt Samples
Two kinds of fermented goat milk were compared in sensory evaluation. Sensory attributes of product are critical indicators of consumer experience. Hence, sensory analysis was conducted to determine whether co-fermentation of LAB and K. marxianus
would affect the organoleptic characteristics of goat yogurt products. Samples were scored for appearance, texture, taste, overall acceptability and goaty flavor. Results are showed in Figure 7
No significant difference was found in appearance and texture based on the scores given by the 12 panelists for GY and GYY. For taste, GYY got higher scores than GY, probably due to the special irritate taste in GYY which caused by carbon dioxide. Carbon dioxide is reported in alcoholic fermentation of the sugars and K. marxianus
lactose metabolism, and it is desirable in certain fermented milk, such as Kefir. Gadaga et al. [39
] investigated yeast in Zimbabwean traditionally fermented milk and found that all of the LAB and yeast cultures produced carbon dioxide as they grew in the UHT milk. One of the LAB and yeast cultures could produce the highest levels of carbon dioxide (approximately 3000–4000 mg kg−1
). In addition, the high titratable acidity of GYY might give GYY a better balance of sugars and acids than GY, leading to the preferred taste of GYY. For goaty flavor, GYY received a lower average score (2.83) than GY (5.25), which indicated that GYY had the lower goaty flavor than GY. GYY was preferred by the panelists due to the higher average score (6.00) of overall acceptability than GY (4.75).
In conjunction with the results of E-nose, GC-MS, and sensory analysis, the aroma compounds in GY and GYY were quite different. The relative contents of three fatty acids were found lower in GYY (caproic = 5.76%, caprylic = 5.74%, capric acid = 5.80%) than GY (caproic = 7.01%, caprylic = 7.01%, capric acid = 6.57%). This result might contribute to the decrease of goaty flavor in GYY. Moreover, plenty of newly formed aroma compounds were detected in GYY, for instance, ethyl acetate (6.05%) with a fruity aroma, isoamyl acetate (5.18%) with the banana flavor, phenethyl alcohol (5.70%) with honey and rose odor and methylnonylketone (4.99%) with a pleasant smell, which were considered to mask the goaty flavor and give GYY a pleasant flavor. Therefore, these results indicated that co-fermentation of LAB and K. marxianus has a positive effect on goat yogurt flavor formation.