Research on the Effect of Simultaneous and Sequential Fermentation with Saccharomyces cerevisiae and Lactobacillus plantarum on Antioxidant Activity and Flavor of Apple Cider

Abstract

The study examined the effect of Lactobacillus plantarum together with Saccharomyces cerevisiae on cider quality through simultaneous and sequential inoculation strategies to evoke malolactic fermentation. The antioxidant activities and flavor compound profiles of apple ciders fermented with mixed cultures of commercial wine yeast (S. cerevisia SY) and autochthonous bacteria (L. plantarum SCFF107 and L. plantarum SCFF200) were assessed. The antioxidant ability results indicated that apple ciders fermented with the simultaneous inoculation method had a higher DPPH radical scavenging rate and total antioxidant capacity, especially for SIL107 cider (simultaneous inoculation with S. cerevisiae SY and L. plantarum SCFF107), which exhibited the highest DPPH free radical scavenging activity (78.14% ± 0.78%) and the highest total antioxidant ability (255.92 ± 7.68 mmol/L). The results showed that ciders produced by mixed inoculation with L. plantarum improved flavor because of their higher contents of volatiles such as esters and higher alcohols and higher contents of non-volatile compounds like organic acids and polyphenols in comparison with the single culture of S. cerevisiae, especially for the simultaneous inoculation method. In addition, irrespective of the inoculation mode, compared to the single culture of cider, L-malic acid degraded dramatically in the presence of L. plantarum during alcoholic fermentation, accompanied by increases in lactic acid. What is more, sensory evaluation results demonstrated that ciders produced by mixed cultures gained higher scores than ciders fermented by the single culture of S. cerevisiae, especially in the simultaneous inoculation mode, in terms of the floral, fruity, and overall acceptability of the cider. Therefore, our results indicated that simultaneous inoculation with L. plantarum was found to compensate for some enological shortages of single S. cerevisiae fermented ciders, which could be a potential strategy to enhance the quality of cider products.

1. Introduction

Gala apple (Malus domestica Borkh) has a sweet and perfumy flavor, differentiating it from other apple cultivars [1]. This apple is very popular among consumers for its unique flavor and taste, and consumer sensory panels almost always gave Gala a very high score on a 9-point hedonic scale [2]. In particular, Gala apples planted in Yanyuan County in Sichuan Province have unique characteristics such as being pollution-free and having higher concentrations of sugar because of the natural ecological environment, high levels of sun exposure, and significant temperature differences between day and night. Nowadays, a large quantity of Gala apples is usually used for fresh consumption. To enrich Gala apple products and meet consumer needs, cider, a value-added product, is one of the essential directions of its processing because it not only preserves the original nutrition and distinct aroma of the fruit but also can meet consumers’ demand for functional foods [3]. However, little research has been reported on the cider fermented with Yanyuan Gala apples.

During fruit wine fermentation, yeasts and bacteria are mainly the critical microorganisms for the production of flavor compounds, such as esters, organic acids, and terpenes. Yeasts are primarily responsible for alcoholic fermentation (AF), in which ethanol and some vital esters are produced [4]. Lactic acid bacteria (LABs) could be used for malolactic fermentation (MLF) and have the ability to reduce acidity, remove residual nutrients, maintain microbial stability, and form aroma profiles, respectively [5]. MLF, which starts immediately after AF, is a secondary biological fermentation mainly evoked by LABs such as Lactobacillus, Oenococcus, Pediococcus, and Leuconostoc [5,6]. Oenococcus oeni, which is the most generally used, is widely employed as a starter culture to conduct MLF [7]. Lactobacillus, on the other hand, has attracted a lot of attention during MLF because they can produce and secrete more flavor-related enzymes than O. oeni. In addition, many reports indicated that Lactobacillus strains could be used as a potential adjunct to yeast to enhance the quality of fruit wines [8,9]. However, little research on screening and application of L. plantarum strains suitable for conducting MLF in the cider fermentation with Gala apple has not been done.

Traditionally, MLF is evoked either by occurring spontaneously in fruit or through intentional inoculation with some microorganisms. Sequential inoculation conducted with MLF could prevent the production of excessive acetic and lactic acid due to heterofermentative sugar metabolism [10]. However, this inoculation mode has its disadvantages; particularly, after AF, yeasts would produce some growth inhibitory metabolites such as ethanol, fatty acids, and SO₂, which would prevent bacterial growth. To mitigate this drawback, the yeast and bacterial cultures can be simultaneously inoculated into fruit juice so as to make the bacteria well acclimatized to the harsh fermentation environment of fruit wine [11]. The simultaneous inoculation method has been successfully employed in some fruit wine fermentations [5,12,13]. In addition, the simultaneous inoculation mode of MLF shortens the fermentation time of fruit wine. However, simultaneous inoculation of yeast and bacteria cultures may result in higher acidity contents or have a negative effect on AF because excessive bacterial growth inhibits the growth and metabolic activities of yeasts [14].

Therefore, this research aimed to investigate MLF in the fermentation of Gala apple cider and to explore the impact of a simultaneous and sequential inoculation strategy conducting MLF on the fermentation performance of cultures of L. plantarum and S. cerevisiae, especially for the effects on antioxidant activities and flavor profiles of Gala apple ciders.

2. Materials and Methods

2.1. Raw Material, Yeast and Bacterial Strains, and Culture Media

Gala apples were purchased from the local supermarket in Yanyuan County, Sichuan Province, China. The commercial wine strain of S. cerevisiae SY (Angel Yeast Co., Ltd., Yichang, China) was cultured with yeast extract, peptone, and glucose (YPD, Aobox, Beijing, China) medium at 28 °C. Two autochthonous L. plantarum strains (SCFF107 and SCFF200) isolated from apples were cultured using De Man, Rogosa, and Sharpe (MRS, Aobox, Beijing, China) medium at 37 °C.

2.2. Apple Cider Making

Apple ciders were made according to previous studies with some modifications [15,16]. The apples were washed and then drained. Subsequently, the apple pulp was cut into small pieces and crushed. Ascorbic acid (0.08% w/v) was immediately added to apple juice to prevent browning. Apple juice was pasteurized (95 °C, 5 min) in the conical flask and subsequently cooled down to room temperature. Before inoculation, the S. cerevisiae strain was cultured in YPD liquid medium in a shaker at a speed of 150 rpm at 28 °C for 24 h, and the L. plantarum strain was incubated in MRS liquid medium at 37 °C for 16 h. S. cerevisiae and L. plantarum were cultured for two generations in an anaerobic environment to make them grow well in oxygen-free conditions, respectively. Afterwards, cells were separated from the liquid medium through centrifugation (4500× g, 4 °C, 10 min). The pellets were washed with sterile saline (0.9% w/v) and centrifuged subsequently as described above, and the elution was performed successively three times. The pellets were subsequently resuspended in apple juice for the subsequent fermentation. All microbial cultures were inoculated at a final density of 10⁷ CFU/mL in 1000 mL of apple juice. Five fermentation tests were subsequently carried out in this study, as follows: (1) single inoculation with the commercial strain S. cerevisiae SY (SA); (2) simultaneous inoculation with S. cerevisiae SY and L. plantarum SCFF107 (SIL107, inoculation with the culture of L. plantarum at 24 h after S. cerevisiae SY inoculation); (3) simultaneous inoculation with S. cerevisiae SY and L. plantarum SCFF200 (SIL200, inoculation with the culture of L. plantarum at 24 h after S. cerevisiae SY inoculation); (4) sequential inoculation with S. cerevisiae SY and L. plantarum SCFF107 (SEL107, inoculation with the culture of L. plantarum on day eight after S. cerevisiae SY inoculation); and (5) sequential inoculation with S. cerevisiae SY and L. plantarum SCFF200 (SEL200, inoculation with the culture of L. plantarum on day eight after S. cerevisiae SY inoculation); Gala apple juice with no inoculation was employed as a control (GL). The inoculation ratio of species in co-fermentation samples was 1:1. The apple juice suspension with single yeast inoculation or mixed inoculation were fermented at 20 °C in darkness for 14 days. At the end of the fermentation, apple ciders were collected from the microbial strains and lees through centrifugation (7000× g, 4 °C, 10 min). The supernatants of apple ciders were stored at −20 °C to prevent light and oxygen interference for further analysis.

2.3. Basic Oenological Analysis

Physical and chemical analyses of the samples were performed on the basis of the method described previously, with some changes [3]. The pH values of apple juice and apple cider were measured with a pH meter (PH-100, Lichen, Shanghai, China). The total acid contents were determined by acid-base titration with 0.1 M NaOH, and the results were expressed as malic acid. The contents of reducing sugar were determined by 3,5-Dinitrosalicylic acid. The alcoholic contents of apple ciders were determined on the basis of the second method in GB 5009.225–2016.

2.4. Determination of Microbial Counts

Before the yeast and bacterial cells were counted, each sample was diluted serially with a sterile solution of sodium chloride (0.9% w/v). The counts of S. cerevisiae cells were performed after plating a 0.1 mL aliquot of each dilution onto PDA plates and incubating successively at 28 °C for 24 h to form visible colonies [17]. A similar approach was employed to determine the L. plantarum cell counts on the MRS agar plate with 0.2 g/L nystatin and cultured in an anaerobic environment at 37 °C for 48 h [5].

2.5. Determination of Antioxidant Activity

The antioxidant capacities of the apple juice and ciders were determined by measuring the total antioxidant activity and DPPH-free radical superoxide anion-reducing power using commercial assay kits purchased from Sangon Biotech (Shanghai, China).

2.6. Volatile Compound Analysis

The volatile component analyses were performed on the basis of the previous method, with some changes [15]. GC-TOF-MS analysis was carried out with an Agilent 7890 gas chromatograph in conjunction with a time-of-flight mass spectrometer (MS). The Agilent DB-5MS capillary column was applied in the system, and the carrier gas was Helium. The injection volume was 1 μL in splitless mode. The front inlet purge flow was 3 mL/min, and the gas flow rate through the column was 1 mL/min. The initial temperature was 50 °C (held for 1 min), subsequently raised to 310 °C at a speed of 10 °C/min, and kept at this temperature for 8 min. The injection, transfer line, and ion source temperatures were 280 °C, 280 °C, and 250 °C, respectively. Electron ionization (electron impact mode at 70 eV) spectra in the m/z range from 50 to 500 were acquired in full-scan mode at 12.5 spectra per second after a solvent delay of 6.25 min. The volatile compounds were determined using semi-quantitative analysis based on the added internal standard (2-Octanol).

2.7. Non-Volatile Compound Analysis

The non-volatile component analyses were performed on the basis of the method described previously, with some modifications [18]. LC-MS/MS analysis was carried out with the UHPLC system (Vanquish, Thermo Fisher Scientific) coupled with a Waters ACQUITY UPLC BEH Amide column (2.1 mm × 100 mm, 1.7 μm) and Q Exactive HFX mass spectrometer (Orbitrap MS, Thermo Fisher Scientific). Two solvents were used to elute: mobile phase A (25 mmol/L ammonia hydroxide and 25 mmol/L ammonium acetate in water) and mobile phase B (acetonitrile). The auto-sampler temperature was kept at 4 °C, and a 2-μL aliquot of samples was injected. The QE HFX MS was applied to get MS/MS spectra in information-dependent acquisition mode with the acquisition software (Xcalibur, Thermo). In this mode, the acquisition software continuously evaluates the full scan MS spectrum. The operating conditions of the electrospray ionization source were applied as follows: Sheath gas flow rate was 30 Arb; aux gas flow rate was 25 Arb; the capillary temperature was 350 °C; full MS resolution was 120,000; MS/MS resolution was 7500; collision energy was 10/30/60 eV in NCE mode; and spray voltage was 3.6 kV (positive) or −3.2 kV (negative).

2.8. Sensory Analysis

Sensory analyses were performed on the basis of the previously described method, with some changes [19]. The sensory properties of apple cider samples were evaluated and scored by 15 trained panelists (students and teachers) with relevant experience and background knowledge. The sensory quality of the cider was evaluated using six attributes: fruity, sweetness, bitterness, flavor, sour, and overall acceptability, and was scored according to the nine-point hedonic scale, where one represented the poor and nine indicated the excellent. About 50 mL of each cider sample was served in wine glasses labeled with random numbers and then evaluated under white light and at room temperature. The sensory quality of each finished cider was assessed by calculating and plotting the average scores of all characters.

2.9. Statistical Analyses

Each experiment was carried out in triplicate, and all data were expressed as means ± standard deviation (SD). Difference analyses between experimental groups were performed with Duncan’s multiple comparison test with IBM SPSS version 26 (SPSS Inc., Chicago, IL, USA), and the level of statistical significance was accepted at at least 5%. Principal component analysis (PCA) and hierarchical cluster analysis (HCA) were conducted with Origin 9.0 software (Hampton, MA, USA).

3. Results

3.1. Dynamic Evolution of Basic Oenological Parameters

The dynamic changes of basic oenological parameters of apple ciders are presented in Figure 1. Acidity is a crucial parameter that has an effect on the unique taste and refreshing traits of fruit wines [20]. As displayed in Figure 1a, the pH values of apple juice during the fermentation process exhibited a downward trend. However, the pH values fluctuated greatly from the 6th day to the 10th day in simultaneously fermented ciders, which may be related to the synthesis and decomposition of some organic acids, such as malic acid and lactic acid [21]. Moreover, the pH values in co-fermented cider were higher than those in a single culture of cider at the end of the fermentation. As displayed in Figure 1b, the changes in total acids were negatively correlated with the changes in pH values. The total acid contents of apple ciders were higher than those of apple juice, which was consistent with previous studies [22,23]. Moreover, the total acid contents of the co-fermentation ciders with L. plantarum were significantly lower than that of the single culture of cider (SA), irrespective of the inoculation method, suggesting that L. plantarum had deacidification ability. Our results were consistent with the previous study [24].

As shown in Figure 1c, the reducing sugar contents began with a descending trend and then stabilized. At the end of fermentation, the reducing sugar contents of sequential fermentation ciders were lower than those of simultaneous fermentation and single-culture ciders, which was attributed to the different sugar consumption capacities of yeast and L. plantarum [16]. As reducing sugars were consumed, the alcohol percentage in all apple ciders increased and remained stable on day 8 (Figure 1d). Compared to the single culture of cider (SA), irrespective of the inoculation method used, mixed cultures of cider had a lower alcohol percentage. Furthermore, simultaneous fermentation ciders had a lower alcohol percentage than sequential fermentation ciders.

3.2. Evolution of Microbial Populations

The dynamic changes in S. cerevisiae and L. plantarum cell counts in different cider samples are shown in Figure 2. S. cerevisiae reached its maximum population of 7.3 × 10⁸ CFU/mL in all ciders on day two and then declined. In the simultaneous inoculation (Figure 2b), compared to single-culture inoculation, S. cerevisiae decreased much more with the addition of L. plantarum on day two, because L. plantarum might consume the nutrients required for yeast or produce yeast growth inhibitors, such as some organic acids [5]. Furthermore, L. plantarum cells increased from day two to day five and then remained stationary, indicating that L. plantarum could be well adapted to apple cider fermentation conditions, which was in agreement with previous findings [21]. In the sequential inoculation (Figure 2c), S. cerevisiae continued declining with adding L. plantarum on day eight, and then remained stationary from day ten, whereas L. plantarum cells increased in the first two days and then remained stable with more L. plantarum cells than S. cerevisiae cells, suggesting that L. plantarum could also be well adapted to the cider environment in sequential inoculation fermentation.

3.3. Evolution of Antioxidant Activity

Apple juice is rich in plenty of active substances, such as organic acids, polyphenols, and flavonoids, which exhibit strong antioxidant properties. Accumulating studies demonstrated that phenolic compounds possessed remarkable antioxidant activity and were used as reducing agents, free-radical scavengers, and singlet oxygen quenchers, because they had the ability to transfer hydrogen atoms or donate electrons to free radicals [25]. In this study, the dynamics of DPPH free radicals, superoxide anion, and total antioxidant capacity in each fermented cider were studied. Overall, mixed culture fermentation, irrespective of the inoculation method, had higher antioxidant activities than single culture fermentation, and antioxidant activity in cider with simultaneous inoculation fermentation was higher than that in cider with sequential inoculation fermentation, especially for SIL107 cider, which exhibited the highest DPPH free radical scavenging rate (78.14% ± 0.78%) and total antioxidant ability (255.92 ± 7.68 mmol/L) at the end of fermentation.

Figure 3a showed that the DPPH free radical scavenging rate of apple ciders co-fermented with S. cerevisiae and L. plantarum was higher than that of apple juice (GL) and the single culture of cider (SA). The DPPH free radical scavenging rate of mixed cultures of cider reached its maximum on day ten after adding L. plantarum because L. plantarum could enhance the utilization of polyphenol compounds [26]. After the second day, the DPPH free radical scavenging rates of the simultaneous inoculation ciders were significantly higher than those of the sequential inoculation ciders during the fermentation process, especially for SIL107 cider, which had the highest DPPH free radical scavenging rate at the end of the fermentation. Moreover, after co-fermentation with L. plantarum, regardless of the inoculation method, the DPPH free radical scavenging abilities of ciders fermented with L. plantarum SCFF107 were higher than those of the ciders fermented with L. plantarum SCFF200, suggesting a significant difference in DPPH free radical scavenging rate in different L. plantarum strains.

Figure 3b displays the dynamics of the total antioxidant capacity of each fermentation sample. Mixed culture fermentation, irrespective of the inoculation method used, had higher total antioxidant ability than apple juice and the single culture of cider, and total antioxidant activities in ciders with simultaneous inoculation fermentation were higher than those in ciders with sequential inoculation fermentation, revealing that L. plantarum also enhanced the total antioxidant abilities of ciders, which was similar to the previous study [27]. Moreover, whether in simultaneous or sequential inoculation fermentation, similar to the DPPH free radical scavenging rate, ciders fermented with L. plantarum SCFF107 had higher total antioxidant abilities than ciders fermented with L. plantarum SCFF200.

3.4. Analysis of Volatiles

The volatile compounds are one of the most crucial attributes of cider for consumer palatability [28]. In this study, a total of 38 volatiles were determined in all samples, including 20 esters, 12 higher alcohols, and six ketones (

Table 1. Volatile compounds of apple juice and ciders produced by pure and mixed culture fermentations.

Number	Compounds	GL	Treatment (n = 3) (μg/L)
			SA	SIL107	SIL200	SEL107	SEL200
	Esters
1	4-hydroxybutyrate	-	434.33 ± 37.88 d	1733.15 ± 60.60 a	1707.32 ± 72.40 a	1422.87 ± 38.86 b	1050.73 ± 14.54 c
2	Isoamyl acetate	1.68 ± 0.59 e	138.48 ± 3.62 d	288.25 ± 9.52 a	217.59 ± 15.96 b	184.03 ± 11.24 c	146.72 ± 5.76 d
3	Methyl trans-cinnamate	-	123.39 ± 10.03 d	829.81 ± 29.42 a	459.11 ± 29.9 c	612.09 ± 7.77 b	570.31 ± 41.22 b
4	Ethyl hexanoate	2.44 ± 0.43 f	802.73 ± 10.26 e	1154.57 ± 68.18 a	1017.18 ± 6.17 b	949.13 ± 35.65 c	873.85 ± 12.94 d
5	Hexyl acetate	22.80 ± 0.88 c	50.76 ± 1.19 b	64.11 ± 1.91 a	63.35 ± 1.00 a	52.14 ± 0.28 b	52.60 ± 0.61 b
6	Dioctyl phthalate	368.91 ± 331.12 d	804.12 ± 58.97 c	1244.64 ± 26.22 ab	839.41 ± 17.64 c	1418.40 ± 8.85 a	1148.51 ± 10.17 b
7	Diethylphosphate	9.43 ± 0.99 c	81.88 ± 8.15 ab	84.43 ± 16.10 ab	95.46 ± 4.30 a	74.60 ± 1.10 b	88.76 ± 3.20 ab
8	Ethyl butanoate	23.11 ± 1.11 f	103.38 ± 1.26 e	164.52 ± 8.45 a	147.86 ± 6.36 b	117.08 ± 6.71 d	136.37 ± 2.97 c
9	2-Isopropylphenyl methylcarbamate	64.47 ± 0.22 b	191.39 ± 3.49 a	184.17 ± 6.12 a	179.56 ± 9.62 a	188.93 ± 15.12 a	193.38 ± 3.13 a
10	Methyl 3-(methylthio)propanoate	4.91 ± 1.13 e	12.23 ± 0.48 d	13.18 ± 0.47 cd	16.86 ± 0.88 a	14.56 ± 0.26 b	13.91 ± 0.23 bc
11	Monoethyl malonic acid	4.41 ± 0.27 e	36.27 ± 3.08 c	53.69 ± 1.93 a	44.73 ± 4.97 b	44.56 ± 0.63 b	25.05 ± 2.28 d
12	D-glucurono-6,3-lactone	94.60 ± 7.38 e	1039.44 ± 21.81 c	1656.37 ± 28.00 a	1142.89 ± 21.33 b	734.44 ± 94.57 d	167.39 ± 14.77 e
13	Phenylphosphoric acid	-	90.61 ± 56.00 bc	250.92 ± 95.84 a	112.21 ± 55.70 b	133.14 ± 40.92 b	111.95 ± 30.92 b
14	Ethyl caprylate	4.65 ± 0.38 e	526.29 ± 14.97 d	838.80 ± 18.93 a	716.03 ± 4.20 b	734.29 ± 4.65 b	658.41 ± 4.89 c
15	Methyl cinnamate	896.57 ± 1.05 f	1786.83 ± 11.91 e	2570.10 ± 100.18 a	2230.46 ± 36.37 c	2469.53 ± 16.42 b	2040.02 ± 12.40 d
16	4-(Trimethylammonio)butanoate	177.56 ± 8.90 c	222.37 ± 3.65 b	264.95 ± 10.27 a	176.14 ± 2.54 c	174.90 ± 3.90 c	123.43 ± 4.90 d
17	Methyl 2-ethyl malonate	359.91 ± 13.87 d	1340.49 ± 25.42 a	967.25 ± 20.82 b	1001.41 ± 32.72 b	601.28 ± 9.93 c	272.83 ± 2.49 e
18	Triscarbamate	9.50 ± 0.09 e	346.91 ± 27.15 d	534.95 ± 11.04 b	674.07 ± 15.60 a	463.11 ± 31.39 c	-
19	Caffeic acid, ethyl ester	83.95 ± 2.84 e	934.28 ± 15.44 d	1253.29 ± 7.49 b	1087.00 ± 6.82 c	1283.01 ± 13.14 a	-
20	Bisphthalate	3.40 ± 0.36 e	990.32 ± 0.15 c	1156.81 ± 27.84 a	1047.93 ± 10.06 b	1148.51 ± 10.17 a	941.49 ± 27.31 d
Ʃ(Sum)		2132.31 ± 320.96 e	10,056.48 ± 52.56 c	15,07.95 ± 376.28 a	12,976.55 ± 173.18 b	12,820.59 ± 28.58 b	8615.71 ± 62.94 d
	Higher alcohols
1	2,3-Butanediol	53.18 ± 24.23 d	166.65 ± 2.29 c	227.62 ± 23.38 b	274.33 ± 27.07 a	215.15 ± 26.69 b	149.51 ± 32.30 c
2	Isobutanol	1.29 ± 0.26 f	49.22 ± 0.06 d	68.49 ± 0.78 a	59.13 ± 0.19 c	66.87 ± 1.47 b	41.74 ± 0.29 e
3	Phenethyl alcohol	3.54 ± 0.09 b	23.11 ± 0.59 b	333.71 ± 247.27 a	40.48 ± 0.26 b	34.52 ± 0.22 b	32.01 ± 0.54 b
4	Glycerin	5.79 ± 0.06 f	402.48 ± 1.01 e	547.90 ± 5.88 a	442.02 ± 0.16 d	484.35 ± 4.11 b	462.35 ± 0.52 c
5	3-Methyl-1-butanol	22.83 ± 2.29 f	3803.68 ± 1.04 e	4184.73 ± 7.69 a	4046.17 ± 4.05 b	3994.67 ± 0.71 c	3666.11 ± 45.21 d
6	2-Amino-3-methyl-1-butanol	-	1528.87 ± 21.93 d	4399.72 ± 150.79 a	1895.32 ± 37.67 b	1479.70 ± 25.45 e	1664.05 ± 90.44 c
7	3-Methylamino-1,2-Propanediol	-	1262.27 ± 15.07 b	2812.11 ± 1287.54 a	1308.75 ± 512.95 b	1468.32 ± 383.83 b	1418.26 ± 650.62 b
8	2-Butyne-1,4-diol	-	299.05 ± 9.34 b	443.61 ± 16.72 a	228.22 ± 15.87 b	444.19 ± 8.17 a	259.65 ± 12.85 b
9	2-Deoxyerythritol	-	118.47 ± 4.63 d	516.08 ± 7.18 a	178.50 ± 5.33 c	182.31 ± 8.74 c	225.93 ± 14.90 b
10	1-Indanol	-	120.35 ± 9.99 d	422.29 ± 5.19 a	216.80 ± 10.72 b	139.04 ± 19.57 cd	166.70 ± 34.37 c
11	Estriol	1128.46 ± 51.22 a	233.33 ± 13.16 e	988.64 ± 9.00 b	204.44 ± 0.52 e	331.82 ± 2.58 c	290.35 ± 4.12 d
12	Diglycerol	54.47 ± 4.26 e	111.86 ± 2.53 d	422.83 ± 18.71 a	129.25 ± 8.82 c	172.68 ± 0.99 b	123.81 ± 2.85 cd
Ʃ(Sum)		1269.56 ± 40.01 c	8119.32 ± 34.57 b	15,367.73 ± 1484.83 a	9023.40 ± 533.74 b	9013.62 ± 418.84 b	8500.48 ± 690.86 b
	Ketones
1	Acetophenone	-	206.66 ± 3.31 d	624.05 ± 7.72 a	308.66 ± 15.25 b	276.75 ± 14.6 c	199.6 ± 6.77 d
2	Dihydroxyacetone	-	1402.92 ± 77.31 e	4545.63 ± 175.26 b	3205.09 ± 20.11 d	5262.09 ± 37.45 a	3713.40 ± 62.40 c
3	1,3-Cyclohexanedione	-	373.97 ± 14.43 c	1251.82 ± 16.19 a	634.82 ± 14.78 b	375.81 ± 5.06 c	392.25 ± 2.28 c
4	Acetol	-	985.30 ± 27.78 c	3246.30 ± 64.58 a	861.95 ± 26.27 d	1215.16 ± 3.96 b	865.50 ± 18.43 d
5	21-Hydroxypregnenolone 2	65.73 ± 4.56 c	75.96 ± 3.41 b	63.73 ± 0.76 c	53.00 ± 3.55 d	64.35 ± 3.32 c	280.41 ± 10.38 a
6	3,4-Dihydroxyphenylacetone	843.71 ± 21.16 c	993.74 ± 19.29 b	1017.23 ± 88.70 b	2794.66 ± 52.82 a	963.30 ± 37.07 b	1001.97 ± 14.58 b
Ʃ(Sum)		909.44 ± 25.54 f	4038.54 ± 57.91 e	10,748.77 ± 324.99 a	7858.17 ± 51.67 c	8157.46 ± 80.42 b	6453.14 ± 94.39 d
Ʃ(Sum: GC-MS)		4311.31 ± 151.29 e	22,214.33 ± 159.23 d	41,424.45 ± 522.29 a	29,858.12 ± 614.05b	29,991.67 ± 227.51 c	23,569.32 ± 223.77 d

Note: “-” means not detected. Data with different letters (a, b, c, d, e, f, g) within each row are significantly different (p < 0.05).

). The total volatile response values of co-fermented ciders were significantly higher than those in the single culture of cider (SA), indicating that L. plantarum improved the production of aroma-creating volatile substances because co-fermentation of S. cerevisiae and L. plantarum contributed to regulating the volatile compounds and the complexity of the cider aroma [21]. Moreover, ciders with simultaneous inoculation fermentation had higher volatile compounds than ciders with sequential inoculation fermentation. In addition, whether in simultaneous or sequential inoculation fermentation, ciders fermented with L. plantarum SCFF107 had higher contents of volatile compounds than ciders fermented with L. plantarum SCFF200. Partcicularly, SIL107 ciders had the highest total volatile compound concentrations (41.42 ± 0.52 mg/L).

The most abundant volatile compounds were esters in the apple juice and finished ciders (Table 1). Esters are volatile compounds considered to have an essential impact on cider flavor and provide pivotal qualities in terms of desired fruity aromas [29,30]. As displayed in Figure 4a, ester concentrations in co-fermented ciders (SIL107, SIL200, and SEL107) were significantly higher than those in apple juice (GL) and single cultures of cider (SA), suggesting that the mixed cultures of cider helped the accumulation of ester components, confirming the previous studies [4,31]. Moreover, ciders fermented with simultaneous inoculation method had higher esters than those fermented with the sequential inoculation method. In addition, irrespective of the inoculation mode, ciders fermented with L. plantarum SCFF107 had higher ester concentrations than ciders fermented with the addition of L. plantarum SCFF200. SIL107 cider, in particular, was characterized by the highest levels of esters (15.31 ± 0.38 mg/L).

Higher alcohols, which were deemed to be one of the most significant precursors of esters, which were conducive to fresh fruity notes, such as 2,3-butanediol and glycerin, were believed to give a pleasant attribute to the aromatic complexity of fruit wines when their concentrations were below 300 mg/L [15,32]. As shown in Figure 4b, the contents of higher alcohols in co-fermented ciders (from 8.50 ± 0. 69 mg/L to 15.37 ± 1.48 mg/L) were higher than those in apple juice (1.27 ± 0.04 mg/L) and single-culture cider (8.12 ± 0.03 mg/L) (Table 1). Moreover, alcohols, together with organic acids, contributed to the production of esters with a pleasant taste. As displayed in Figure 4c, the production of ketones increased considerably in co-fermented ciders compared to the single culture of cider (SA), especially for SIL107 cider, which had the highest ketone content (10.75 ± 0.32 mg/L). Consequently, ciders fermented with mixed cultures not only increased esters but also enhanced higher alcohols and ketones, especially with the simultaneous inoculation method.

To distinguish volatiles in apple juice, the single culture of cider, and mixed cultures of ciders, PCA analysis was conducted. The first and second explained 61.3% (PC1) and 19.1% (PC2) of the total variation, respectively. The scatter plot displayed that apple juice and cider samples were separated from each other (Figure 4d). Apple juice (GL) was positioned in the second quadrant alone, while control cider (SA) and SEL200 were positioned in the third quadrant. SIL107 cider was in the first quadrant, while SEL107 and SIL200 ciders were in the fourth quadrant.

3.5. Analysis of Non-Volatiles

The polyphenols and organic acids in fruits are vital properties because they display positive impacts on a variety of physiological processes in human health, such as anti-inflammatory properties and a reduction of cancer risk [33,34]. A total of 76 non-volatile compounds were determined in all samples, including 29 organic acids, 39 polyphenols, and eight terpenoids (

Table 2. Non-volatile compounds of apple juice and apple ciders produced by mono- and co-fermentations.

Number	Compounds	GL	Treatment (n = 3) (μg/L)
			SA	SIL107	SIL200	SEL107	SEL200
	Organic acids
1	Pyruvic acid	1643.22 ± 403.03 e	34,544.94 ± 1802.35 d	65,988.87 ± 2956.05 a	42,247.69 ± 3048.87 c	51,645.63 ± 1133.67 b	37,289.65 ± 1349.1 d
2	Glycolic acid	4682.42 ± 316.75 f	11,768.34 ± 763.92 c	19,482.25 ± 261.03 b	7502.83 ± 414.52 e	23,995.42 ± 1182.98 a	10,445.44 ± 54.93 d
3	2-Ketobutyric acid	-	1471.06 ± 33.79 b	2923.11 ± 45.68 a	853.64 ± 39.00 d	1143.15 ± 20.33 c	1214.12 ± 122.55 c
4	2-Keto-Isovaleric acid 1	2669.08 ± 185.05a	-	1649.82 ± 145.71 b	700.35 ± 91.75 d	944.51 ± 35.40 c	1049.31 ± 98.08 c
5	2-Hydroxybutanoic acid	2039.9 5± 49.50 e	3437.46 ± 73.10 c	5471.35 ± 57.65 a	3368.03 ± 162.31 c	4570.30 ± 236.40 b	2646.51 ± 265.04 d
6	2-Ketovaleric acid	-	355.72 ± 17.59 e	956.80 ± 13.54 a	750.99 ± 25.47 b	467.63 ± 20.07 c	384.60 ± 52.33 d
7	Malonic acid	427.89 ± 501.62 b	6390.49 ± 1152.74 ab	10,176.54 ± 6317.14 a	5247.16 ± 1941.62 ab	7472.61 ± 2390.47 ab	7479.89 ± 5415.91 ab
8	Maleic acid	520.07 ± 450.58 c	1030.07 ± 32.18 b	3358.77 ± 297.45 a	1072.22 ± 56.77 b	1318.69 ± 7.18 b	1156.80 ± 18.62 b
9	Succinic acid	763.15 ± 29.46 e	1634.35 ± 34.82 b	2806.17 ± 36.80 a	51.90 ± 16.60 f	1252.58 ± 38.22 d	1399.44 ± 33.04 c
10	Fumaric acid	437.32 ± 31.09 e	5844.83 ± 328.14 d	16,132.72 ± 527.39 a	5830.70 ± 522.47 d	8256.59 ± 360.08 c	9765.62 ± 119.63 b
11	Tartronic acid	182.08 ± 14.71d	832.18 ± 20.17f	1111.95 ± 30.39b	755.57 ± 19.35e	1346.42 ± 42.58a	1047.37 ± 21.83c
12	Citric acid	-	429.83 ± 0.99 d	3409.68 ± 50.03 b	1616.14 ± 146.73 e	821.13 ± 9.98 a	1882.10 ± 65.29 c
13	Glucuronic acid	-	11,245.81 ± 978.41 e	27,205.79 ± 1485.87 b	36,176.00 ± 1445.20 a	17,370.89 ± 796.59 d	20,830.75 ± 310.45 c
14	Galactonic acid	1081.12 ± 173.97 f	3556.68 ± 248.23 d	21,015.36 ± 340.93 a	6100.63 ± 764.19 e	17,181.97 ± 168.89 b	7055.90 ± 677.71 c
15	Gluconic acid	-	287.56 ± 42.40 c	119.33 ± 7.08 d	569.81 ± 15.64 a	144.04 ± 8.50 d	455.60 ± 19.50 b
16	3,4-Dihydroxycinnamic acid	-	18,195.70 ± 544.37 cd	65,767.89 ± 3253.46 a	19,246.32 ± 586.68 bc	21,507.88 ± 1329.01 b	16,399.49 ± 357.20 d
17	Lactobionic Acid	24.78 ± 5.77 e	157.11 ± 26.62 c	735.45 ± 14.84 a	157.03 ± 25.44 c	364.76 ± 6.89 b	111.01 ± 3.68 d
18	PyrrolidonecArboxylic acid	7933.47 ± 753.72 a	1060.88 ± 61.77 d	1939.55 ± 21.47 c	2306.35 ± 77.21 ab	2240.34 ± 67.65 ab	2557.59 ± 152.64 b
19	Vanillic acid		3949.58 ± 164.38 c	4803.67 ± 219.31 a	3633.50 ± 74.49 d	4421.21 ± 209.10 b	2743.35 ± 54.80 e
20	Daucic acid	-	627.97 ± 26.30 c	911.26 ± 8.14 a	549.61 ± 27.06 d	656.01 ± 6.88 c	770.45 ± 38.57 b
21	cis-Aconitic acid	312.34 ± 18.21 f	10,222.02 ± 138.27 e	31,658.31 ± 326.97 a	19,734.22 ± 280.34 c	20,820.07 ± 687.71 b	11,128.07 ± 131.68 d
22	Phenylpyruvic acid	264.77 ± 14.56 e	4014.22 ± 163.71 c	5401.94 ± 118.88 a	4343.76 ± 177.75 b	2174.58 ± 35.33 d	2211.44 ± 119.28 d
23	L-Malic acid	343.57 ± 47.41 a	277.17 ± 11.72 b	40.75 ± 40.36 e	89.45 ± 8.16 cd	118.91 ± 21.87 c	84.18 ± 1.96 cd
24	L-Lactic acid	1135.49 ± 115.13 f	28,972.88 ± 497.55 e	76,585.35 ± 1348.07 a	54,613.28 ± 2720.42 c	67,019.63 ± 1113.69 b	48,011.79 ± 4721.11 d
25	D-Malic acid	342.52 ± 23.64 e	967.27 ± 11.31 d	1076.49 ± 73.14 c	1023.7 ± 58.99 cd	1175.94 ± 86.57 b	1662.27 ± 26.25 a
26	2-Furoic acid	166.08 ± 6.66 d	834.71 ± 2.64 c	999.41 ± 64.44 b	1121.64 ± 68.71 ab	1186.84 ± 102.98 a	1021.07 ± 139.13 b
27	2-Aminoacrylic acid	13,729.5 ± 1250.61 a	471.77 ± 25.60 b	532.23 ± 9.28 b	489.64 ± 24.05 b	338.04 ± 7.42 b	553.41 ± 21.59 b
28	2-Methyl-3-Hydroxybutyric acid	1573.30 ± 390.68 c	1764.95 ± 107.63 c	4121.93 ± 117.92 a	2185.38 ± 122.29 b	2130.52 ± 58.63 b	2224.56 ± 139.98 b
29	5-Aminolevulinic acid	359.20 ± 14.68 f	724.47 ± 13.67 d	1748.97 ± 27.14 a	656.44 ± 35.02 e	934.22 ± 8.85 b	794.13 ± 4.38 c
Ʃ(Sum)		40631.33 ± 2010.48 f	154,833.6 ± 2377.27 e	378,368.09 ± 10396.61 a	222,993.98 ± 5596.48 c	263,020.50 ± 2066.40 b	194,375.91 ± 4694.26 d
	Polyphenols
1	Epicatechin	138.37 ± 6.66 f	29,012.60 ± 527.35 e	62154.47 ± 808.11 a	55728.44 ± 1522.04 b	42622.34 ± 1829.52 c	33762.44 ± 2022.90 d
2	Catechin	1791.49 ± 15.50 a	749.43 ± 40.98 c	922.09 ± 13.10 b	525.76 ± 27.42 d	351.95 ± 17.41 e	238.26 ± 19.05 f
3	Ptelatoside B	132.21 ± 8.11 e	843.49 ± 25.56 b	854.88 ± 16.46 b	1096.14 ± 32.68 a	378.16 ± 13.04 c	309.31 ± 8.14 d
4	Quercetin	36,602.35 ± 117.15 a	7768.82 ± 171.04 c	26,310.33 ± 435.00 b	6612.57 ± 274.95 d	15,974.51 ± 188.12 e	5508.39 ± 257.74 f
5	Eugenol	118.37 ± 0.83 f	1649.13 ± 26.13 c	2886.94 ± 91.05 a	1542.27 ± 42.66 d	1788.13 ± 65.06 b	1149.86 ± 25.34 e
6	Cinnamaldehyde	11.72 ± 10.31 c	2067.41 ± 19.25 b	3019.70 ± 81.41 a	2192.72 ± 38.53 b	2892.84 ± 545.90 a	1880.87 ± 4.29 b
7	(-)-Epiafzelechin	99.78 ± 15.87 e	942.86 ± 20.25 c	1146.97 ± 14.58 a	1004.60 ± 41.05 b	922.55 ± 11.33 c	711.65 ± 61.52 d
8	(-)-Catechin	19.68 ± 8.16 e	288.55 ± 3.36 c	463.75 ± 25.69 a	324.54 ± 21.94 b	233.74 ± 10.40 d	281.37 ± 15.45 c
9	Hyperoside	11.18 ± 0.59 f	69.67 ± 4.55 e	121.14 ± 21.95 d	172.81 ± 12.55 c	207.78 ± 3.37 b	294.36 ± 7.12 a
10	Hesperidin	92.91 ± 1.97 e	3282.23 ± 29.48 d	4641.71 ± 121.51 a	4191.15 ± 163.05 b	3860.34 ± 30.78 c	3405.00 ± 39.70 d
11	Procyanidin B2	231.43 ± 19.18 a	4.26 ± 0.35 c	82.49 ± 7.15 b	5.65 ± 0.07 c	7.23 ± 0.42 c	5.63 ± 0.33 c
12	Phloretin	219.09 ± 6.21 e	654.06 ± 31.86 c	859.18 ± 24.19 a	757.28 ± 20.78 b	455.00 ± 11.52 d	486.41 ± 22.19 d
13	Isorhamnetin	2.13 ± 3.68 e	70.83 ± 4.05 d	208.07 ± 0.80 a	105.62 ± 4.11 b	86.46 ± 9.43 d	88.63 ± 7.14 d
14	Quercetin-3-O-sophoroside	252.36 ± 26.56 a	5.17 ± 0.60 b	8.56 ± 0.24 b	3.36 ± 0.43 b	2.74 ± 0.12 b	8.02 ± 0.49 b
15	Lutein	178.85 ± 6.60 e	354.35 ± 17.01 d	462.54 ± 26.66 c	516.53 ± 14.88 a	489.08 ± 1.36 b	440.70 ± 1.70 c
16	3-O-Caffeoyl-4-O-methylquinic acid	119.06 ± 8.52 e	316.22 ± 3.97 c	530.25 ± 3.53 a	431.13 ± 5.00 b	317.87 ± 15.44 c	276.09 ± 1.74 d
17	Vanilloside	14.45 ± 1.55 d	378.17 ± 14.39 c	639.13 ± 19.65 a	455.20 ± 29.84 b	437.92 ± 6.54 b	353.95 ± 10.92 c
18	Isoquercitrin	211.78 ± 7.65 e	527.81 ± 26.90 c	584.29 ± 12.80 b	530.25 ± 17.56 c	615.02 ± 16.60 a	396.14 ± 2.04 d
19	Curcumin I	180.07 ± 8.03 bc	192.57 ± 6.11 b	226.54 ± 24.03 a	171.73 ± 6.45 bc	192.49 ± 3.71 b	163.42 ± 1.37 c
20	3,4-Dihydroxycinnamic acid	3166.35 ± 11.72 d	2652.23 ± 190.40 e	4715.93 ± 211.34 a	4064.15 ± 30.78 b	3838.03 ± 8.89 c	1469.48 ± 116.02 f
21	Sinapic acid	131.03 ± 6.70 d	163.29 ± 0.66 c	429.03 ± 8.14 a	130.06 ± 6.14 d	215.13 ± 3.10 b	23.33 ± 1.42 e
22	Ferulic acid	19.24 ± 4.98 a	18.74 ± 0.83 b	14.52 ± 2.70 ab	2.60 ± 4.50 c	11.71 ± 4.17 b	0.01 ± 0.00 c
23	1,5-Dicaffeoylquinic acid	8.65 ± 0.47 a	9.24 ± 1.69 a	7.91 ± 1.54 ab	7.21 ± 0.93 ab	6.01 ± 0.97 b	0.10 ± 0.15 c
24	3,4-Dihydroxybenzaldehyde	67.19 ± 4.59 c	79.10 ± 4.87 b	106.11 ± 0.60 a	60.83 ± 3.37 d	58.01 ± 1.55 d	55.65 ± 2.56 d
25	Phlorizin	22,867.47 ± 183.84 f	30,977.57 ± 176.39 c	39,529.56 ± 371.01 a	32,671.37 ± 209.63 b	25,666.46 ± 309.77 e	30,017.00 ± 2.56 d
26	( + )-Catechin	661.55 ± 27.93 d	625.79 ± 6.22 d	887.70 ± 45.31 c	1330.51 ± 32.38 a	1179.10 ± 0.92 b	17.54 ± 1.13 e
27	Bergapten	480.31 ± 11.58 e	2508.01 ± 1.85d	6138.47 ± 21.30 c	15,134.16 ± 13.60 c	5186.70 ± 45.03 c	28,092.95 ± 1549.78 a
28	3,4-Dihydroxybenzoic acid	117.92 ± 6.93 b	90.59 ± 8.51 c	192.99 ± 5.65 a	80.24 ± 12.41 c	106.60 ± 0.30 b	8.81 ± 0.46 d
29	Genistein	104.41 ± 3.64 a	107.62 ± 10.69 a	123.38 ± 92.30 a	108.50 ± 0.36 a	86.12 ± 4.78 a	0.01 ± 0.00b
30	Gallic acid	7160.50 ± 393.28 c	8244.61 ± 57.80 b	9439.28 ± 211.72 a	8233.66 ± 336.09 b	7565.90 ± 182.82 c	5015.77 ± 1.00d
31	Succinate semialdehyde	-	4475.85 ± 63.88 c	11453.38 ± 304.85 a	5399.67 ± 337.68 b	5161.43 ± 63.86 b	4525.48 ± 222.44 c
32	Quinic acid	-	51,940.93 ± 244.52 d	83,310.55 ± 1575.54 a	67,309.36 ± 785.89 b	62,524.53 ± 466.61 c	51,833.43 ± 265.98 d
33	Caffeic acid	-	352.40 ± 41.62 e	1363.67 ± 64.91 a	521.05 ± 5.52 c	807.39 ± 6.19 b	443.16 ± 3.23 d
34	Neohesperidin	-	60.38 ± 3.89 c	420.93 ± 14.70 a	-	116.02 ± 4.49 b	-
35	Epigallocatechin	-	64.17 ± 2.89 d	54.42 ± 2.69 b	73.01 ± 1.15 a	45.26 ± 1.91 c	53.63 ± 0.10 b
36	Chlorogenic Acid	537.27 ± 16.86 f	1030.01 ± 14.68 e	4738.72 ± 11.54 a	1819.99 ± 16.85 d	2077.14 ± 13.51 b	1967.71 ± 33.55 c
37	Naringin	5611.94 ± 212.36 a	657.26 ± 37.78 e	2856.04 ± 119.23 b	941.61 ± 7.86 d	1834.92 ± 8.68 c	752.68 ± 10.75 e
38	Trans-Ferulic acid	3532.12 ± 116.74 a	855.91 ± 18.32 b	1914.69 ± 50.29 c	961.64 ± 27.19 c	879.13 ± 52.76 c	916.22 ± 53.37 c
39	3,5-Dihydroxybenzaldehyde	1387.60 ± 118.06 f	2239.00 ± 45.79 e	7432.99 ± 142.22 b	4291.46 ± 55.72 d	7770.18 ± 123.72 a	5673.13 ± 11.63 c
Ʃ(Sum)		86,280.84 ± 566.02 f	156,330.35 ± 784.93 e	281,253.29 ± 1384.79 a	219,508.87 ± 1854.71 b	196,971.89 ± 2843.26 c	180,626.58 ± 3008.34 d
	Terpenoids
1	Ginkgolide j	1.40 ± 0.99 e	197.94 ± 13.15 c	240.43 ± 9.01 c	117.71 ± 8.53 d	426.54 ± 2.80 b	489.18 ± 56.05 a
2	Xanthoxylin	46.74 ± 1.87 e	2049.57 ± 24.76 c	2861.51 ± 109.67 a	2371.83 ± 3.06 d	2937.94 ± 18.10 a	2616.75 ± 22.22 b
3	(-)-Dihydrocarveol	34.99 ± 10.57 d	102.58 ± 20.08 bc	402.47 ± 15.18 a	84.45 ± 2.80 c	118.09 ± 5.76 b	87.71 ± 5.74 c
4	1,4-Cyclohexanedione 2	1330.41 ± 24.34 f	1611.56 ± 168.71 cd	1737.61 ± 10.01 c	1985.92 ± 10.16 b	1513.64 ± 17.81 de	2492.55 ± 221.21 a
5	Celastrol	354.89 ± 25.20 a	85.80 ± 10.11 b	57.38 ± 2.98 c	74.54 ± 3.05 ab	82.52 ± 5.50 b	74.57 ± 2.18 ab
6	Millefin	287.32 ± 4.06 a	143.15 ± 10.68 f	167.81 ± 2.67 b	148.85 ± 6.84 de	155.55 ± 2.57 cd	164.90 ± 3.72 bc
7	Perillyl acetate	276.15 ± 26.68 c	2358.09 ± 107.42 b	3668.55 ± 133.02 a	3567.11 ± 43.51 a	2269.88 ± 9.63 b	2385.89 ± 9.65 b
8	Osmundalactone	20765.76 ± 164.82 a	8122.54 ± 301.35 c	8063.08 ± 13.24 c	8709.71 ± 165.83 b	7396.50 ± 353.08 d	7604.66 ± 295.83 d
Ʃ(Sum)		23,097.65 ± 134.86 a	14,671.22 ± 155.73 d	17,198.83 ± 74.01 b	17,060.12 ± 126.10 b	14,900.66 ± 367.71 d	15,916.21 ± 143.33 c
Ʃ(Sum: LC-MS)		150,009.83 ± 2411.58 f	325,835.16 ± 2377.11 e	676,820.21 ± 9550.59 a	459,562.96 ± 4288.97 c	474,893.06 ± 1788.72 b	390,918.69 ± 7542.29 d

Note: “-” means not detected. Data with different letters (a, b, c, d, e, f) within each row are significantly different (p < 0.05).

). The total non-volatile components of co-fermented ciders were significantly higher than those in apple juice (GL) and cider fermented with the single culture of S. cerevisiae (SA). Moreover, whether in simultaneous or sequential fermentation, ciders with L. plantarum SCFF107 had higher contents of non-volatile compounds than ciders with L. plantarum SCFF200. In specific, SIL107 ciders had the highest total non-volatile compound concentrations (676.82 ± 9.55 mg/L).

Organic acids can make a difference in sensory perception and affect flavor balancing by regulating pH, stability, and comprehensive quality of the fruit wine [35,36]. In this study, heatmap analysis showed that organic acids in apple juice and ciders could be mainly assigned to two clusters (Figure 5a). Components in cluster I included six organic acids with higher concentrations in apple juice than in ciders, such as pyrrolidone carboxylic acid and L-malic acid. Cluster II had 23 lower levels of organic acids in comparison with apple cider, such as L-lactic acid. Many studies have shown that high levels of L-malic acid in apple ciders leads to a harsh taste and unpleasant flavor, whereas L-lactic acid could improve sensory quality by transforming L-malic acid into L-lactic acid with the help of the malolactic enzyme. In our study, the L-malic acid content was significantly reduced after fermentation, especially for simultaneous inoculation fermentation with the addition of L. plantarum, suggesting that mixed microbial fermentation with L. plantarum had a higher L-malic acid degradation capability than the single culture of S. cerevisiae (Table 2, Figure 5a), which was consistent with the previous study [37]. At the same time, L. plantarum could enhance the safety of the fermentation environment and contribute to the pleasant sensory characteristics of ciders [38]. Compared to the single culture of S. cerevisiae, mixed microbial fermentation ciders had higher contents of glucuronic acid, which could bind itself with toxic metabolites or waste products, keep them water-soluble, and discharge them from the body through excreting urine [39,40]. In addition, the results displayed that pyruvic acid and fumaric acid increased significantly after fermentation, especially for mixed microbial fermentation with L. plantarum, which may be related to the conversion of malic acid, because it is the carbon source for yeast to convert pyruvic acid, fumaric acid, and other compounds [41]. Moreover, high concentrations of pyruvic acid and lactic acid had a positive effect on color stability and the soft perception of fruit wines [42].

Polyphenols had a significant effect on both cider quality and health-promoting properties [34,43]. Previous studies revealed that apple ciders fermented with the addition of L. plantarum were rich in polyphenol content and had higher antioxidant activity [28,44]. As shown in Table 2, polyphenol concentrations in GL and SA samples were lower than those in mixed microbial fermentation ciders, especially for SIL107 cider, which had the highest level of polyphenols (281.25 ± 1.38 mg/L). Heatmap cluster analysis showed decreased and increased polyphenols after the fermentation of apple juice (Figure 5b). Especially, the contents of epicatechin, (-)-catechin, and chlorogenic acid increased significantly after adding L. plantarum, and they were considered to be the essential active substances to enhance antioxidant capacities and reduce lipids [45,46]. It may be that the hydrolase produced by L. plantarum hydrolyzed complex polyphenols into simpler forms and enhanced the antioxidant capacity of ciders [47]. In addition, compared to apple juice and control cider (SA), mixed microbial fermentation with L. plantarum increased some terpenoids in apple ciders, such as xanthoxylin and perillyl acetate.

For further differentiation of non-volatiles in apple juice, single-culture fermented cider, and co-culture fermented ciders, PCA analysis was conducted. The first and second accounted for 58.3% (PC1) and 22.3% (PC2) of the total variation, respectively. The scatter plot displayed that apple juice and cider samples were separated from each other (Figure 5c). Apple juice (GL) and the control cider (SA) were positioned in the second and third quadrants, respectively. SEL107 and SIL200 were positioned in the fourth quadrant, while SIL107 cider was in the first quadrant alone. SEL200 ciders were on edge between the third and fourth quadrants in the negative part of PC2.

3.6. Sensory Evaluation

The sensory evaluation results of the finished ciders were displayed in Figure 6. Compared to single yeast fermentation, the sensory scores of ciders fermented with mixed cultures were much higher. More importantly, the floral, fruity, sweet, and overall acceptability in ciders of mixed cultures were significantly improved, revealing that the addition of L. plantarum had a positive effect on the floral, fruity, and overall acceptability of ciders, which was consistent with the previous reports [48,49,50]. Moreover, ciders fermented with the simultaneous inoculation method had higher sensory scores than ciders fermented with the sequential inoculation method. In particular, SIL107 cider had the highest sensory scores due to its high content of esters or higher alcohols, which could bring a fruity and creamy aroma to ciders. Furthermore, sensory evaluation displayed ciders with L. plantarum SCFF107 had higher sensory scores than ciders with L. plantarum SCFF200, whether in simultaneous or sequential fermentation, demonstrating that L. plantarum at the strain level is also an essential factor influencing the sensory quality of ciders.

4. Conclusions

Appropriate L. plantarum strain selection and inoculation methods can directly affect the quality of cider. This study investigated the effects of co-fermentation of S. cerevisiae and L. plantarum using different inoculation methods on the cider quality, including basic oenological parameters, antioxidant activities, volatile compound profiles, non-volatile compound profiles, and sensory qualities. The co-inoculation of two species improved the antioxidant abilities of apple ciders compared to those generated by just one culture of S. cerevisiae, especially for the simultaneous inoculation method. Moreover, compared to cider fermented with the single culture of S. cerevisiae, the ciders fermented with co-fermentation of S. cerevisiae and L. plantarum, irrespective of the inoculation method, had higher concentrations of volatile and non-volatile compounds such as esters, higher alcohols, and polyphenols, and exhibited prominent tastes such as floral and fruity, as well as overall acceptability, especially for the simultaneous inoculation method used. Hence, the results collectively indicated that simultaneous inoculation employing S. cerevisiae and L. plantarum provided a practical approach to enhance the comprehensive quality of apple cider. Our results also laid the foundation for further investigation to explore suitable L. plantarum strains for cider-making. However, the interactions between S. cerevisiae and L. plantarum during the fermentation process are complex and therefore need further studies.