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Article

Impact of Heat Treatment on Hard Cider Enriched with Cryo-Concentrated Apple Must: Microbiological Profile, Functional Properties, and Storage Stability

by
Matheus de Melo Carraro
1,
Isabela Maria Macedo Simon Sola
1,
Raul Dias Moreira dos Santos
2,
Ivo Mottin Demiate
1,2,
Aline Alberti
1,2 and
Alessandro Nogueira
1,2,*
1
Graduate Program in Food Science and Technology, State University of Ponta Grossa (UEPG), Av. Carlos Cavalcanti 4748, Ponta Grossa 84030-900, PR, Brazil
2
Department of Food Engineering, State University of Ponta Grossa (UEPG), Av. Carlos Cavalcanti 4748, Ponta Grossa 84030-900, PR, Brazil
*
Author to whom correspondence should be addressed.
Fermentation 2025, 11(4), 188; https://doi.org/10.3390/fermentation11040188
Submission received: 18 March 2025 / Revised: 31 March 2025 / Accepted: 31 March 2025 / Published: 2 April 2025
(This article belongs to the Special Issue Lactic Acid Bacteria Metabolism)

Abstract

:
This study evaluated the impact of heat treatment on the microbiological, chemical, and functional properties of hard cider enriched with cryo-concentrate over 180 days of storage. The experimental protocol for the hard cider was assessed under three conditions: room temperature (18–23 °C, CA), refrigeration (7–8 °C, CR), and pasteurization at 60 °C for 15 min (P60) and 80 °C for 15 min (P80). The heat treatment employed was mild to preserve the hard cider’s quality. Microbiological results confirmed proper processing conditions. Pasteurization reduced the initial populations of molds and yeasts by 92.9% (P80) and 83.3% (P60), while lactic acid bacteria decreased by over 99.0%. Microbial counts in P60 and P80 continued to decline during storage. Sugar content was the main indicator of instability in P60, particularly at 60 days. Both P60 and P80 ciders exhibited similar reductions in antioxidant activity, with DPPH showing a reduction of 43–45% and ABTS exhibiting a decrease of 50–51%. Additionally, a twofold increase in color intensity (darkening) was observed during storage in heat-treated samples. These findings demonstrate that pasteurization at 80 °C for 15 min effectively extends the shelf life of hard cider with cryo-concentrate to six months at room temperature, offering a practical solution for commercial production.

1. Introduction

Cider, or hard cider, is a sparkling or still beverage obtained by the total or partial alcoholic fermentation of apple musts (dessert and industrial varieties) from freshly pressed apples, reconstituted apple juice concentrates, and a mixture of apple musts [1,2]. Alcohol content can range from less than 0.5% in non-alcoholic ciders to 13.0% in ice ciders. Apple varieties and cider processing technology vary between countries, regions, and producers worldwide. This affects the diversity of sensory attributes such as aroma, color, haze, acidity, astringency, sweetness, carbonation, and foam [3,4].
However, these beverages can have low-intensity attributes such as sweetness, bitterness/astringency, acidity, aroma, and color, which affect their sensory quality. In addition, the compounds responsible for these attributes are present in low concentrations and vary between harvests due to climate and agronomic operations, increasing the probability of fluctuations in sensory quality [5]. In these cases, enriching cider with a cryo-concentrated apple must becomes an interesting strategy to intensify sensory attributes. On the other hand, adding sugars and other nutrients makes hard cider microbiologically unstable.
Pathogenic bacteria in unpasteurized apple juices or apples must include Escherichia coli O157:H7, Salmonella spp., Listeria monocytogenes, and Cryptosporidium parvum. However, during the first five days of alcoholic fermentation, a reduction of 5–7 log CFU/mL for each pathogen can be observed [6]. On the other hand, hard cider can be sweetened with unpasteurized apple must to produce fresh, natural, and sweetened alcoholic beverages. Food-borne pathogens can be introduced from unpasteurized apple must into alcoholic beverages through this back-sugaring process. Although foodborne pathogens generally do not survive in low pH conditions or a high alcohol environment, the extinction of these pathogens has not been established to guarantee the microbiological safety of the products [7].
New technologies are being evaluated to ensure the safety of cider for consumption. Non-thermal methods include high pressure, UV irradiation, ultrasound, and pulsed electric fields [8,9]. These methods have been tested for their potential use in reducing the number of pathogens in apple juice/apple must. Although some methods have been approved by the Food and Drug Administration (FDA), most still have limitations in commercial applications with efficacy.
Thermal processing is the most cost-effective method for ensuring the microbial safety of apple juice or apple cider. However, thermal processing can result in physicochemical changes that can cause negative sensory modifications and reduce the bioavailability of valuable antioxidants in the hard cider. Reducing the risk of microbial contamination without compromising quality attributes is essential. The optimization of processing conditions is therefore key to balancing these aspects.
The pasteurization process is measured in terms of pasteurization units (PUs), defined as the minutes a product is held at 60 °C [10]. There is no cider industry standard regarding enough PUs. Some cider makers have reported using 10–25 PUs, like those used in breweries. However, some sources report <1 and others report 60 PUs for sufficient reductions of microorganisms [11]. PUs can vary depending on the style, purpose, and chemical composition of alcoholic beverages.
The hard cider industry needs to ensure the quality of its products, including long-term quality, during storage. The heat treatment of hard cider with the addition of cryo-concentrated unpasteurized apple must to improve its functional and sensory quality has not been studied. This cryo-concentrate makes the cider much more susceptible to microbiological and chemical alterations. Therefore, this study aimed to assess the impact of different heat treatments on the microbial profile, chemical composition, and bioactive compounds of a hard cider enriched with cryo-concentrated apple must over 180 days of storage.

2. Materials and Methods

2.1. Material

Experiments were conducted using commercial apples of the Gala and Granny Smith varieties (90 kg each), with a ripeness level ranging from 4 to 5 for both cultivars according to Reid et al. [12].
Modified Salmonella–Shigella (SS) agar (K25-610042, Kasvi, Pinhais, PR, BR), xylose-lysine deoxycholate (XLD) agar (K25-610060, Kasvi, Pinhais, PR, BR), Brilliant Green (VB) agar (K25-610009, Kasvi, Pinhais, PR, BR), tetrationate base broth (TT) (7241A, Acumedia®, Neogen®, USA), Rappaport Vassiliadis broth (RVS) (AG-6016, HiMedia®, India) and lactose broth (K25-611202, Kasvi, Pinhais, PR, BR) were used for the detection of Salmonella spp. Lauryl sulfate tryptose (LST) broth (7142A, Acumedia®, Neogen®, USA) was used to detect thermotolerant coliforms and Escherichia coli. Total fungi and yeasts were enumerated on potato dextrose agar (K25-1022, Kasvi, Pinhais, PR, BR) acidified with tartaric acid, and lactic acid bacteria were enumerated on MRS agar (Kasvi, Pinhais, PR, BR).The reagents TPTZ (2,4,6-tri(2-pyridyl)-s-triazine); DPPH (2,2-diphenyl-2-picrylhydrazyl); ABTS (2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid); and standard Trolox (6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid) (97% pure) were purchased from Sigma-Aldrich (Steinheim, Germany). Acetic acid (≥99.7%) and acetonitrile (99.9%) were purchased from J. T. Baker (Phillipsburg, NJ, USA). Ethanol (99.5%) was purchased from Anidrol (Diadema, SP, BR). The standards of the following phenolic compounds were used: caffeic acid (98%), (+)-catechin (98%), (−)-epicatechin (98%), procyanidin B1 (98%), procyanidin B2 (98%), chlorogenic acid (95%), p-coumaric acid (98%); phloridzin (99%), quercetin-3-D-galactoside (hyperoside) (97%), quercetin-3-β-D-glucoside (isoquercetin) (90%), quercetin-3-O-rhamnoside (quercitrin) (78%), quercetin-3-rutinoside (rutin) (94%), and quercetin-O-α-L-arabinofuranoside (avicularin) (purity ≥ 90%). These were purchased from Sigma-Aldrich (Steinheim, Germany). Standards for sugar analysis were as follows: D-glucose and sucrose (99%) (Sigma-Aldrich, Steinheim, Germany) and fructose and glycerol (99%) (Merck, Darmstadt, Germany). Ultrapure water (type 1) was used (Millipore, SP, BR). The culture media and reagents used were of analytical grade.

2.2. Methods

2.2.1. Apple Juice Processing

The apple varieties were processed separately. The cultivars were selected based on their distinct characteristics: Gala, which accounts for 67% of apple production in Brazil, and Granny Smith, known for its high acidity that enhances the freshness and natural acidity of cider. The fruits were carefully selected, weighed, and sanitized by immersion in sodium hypochlorite (150 mg/L for 20 min at ± 4 °C) followed by rinsing in potable water. The fruits were crushed and pressed (AGM Maquinas, Bento Gonçalves, RS, BR) at 294 kPa for 5 min, resulting in the extraction of integral apple must. The average physical yield (ηp) was 72.7%. The apple musts were depectinized using a pectinolytic enzyme (Pectinex® Ultra Clear, LNF Bento Gonçalves, RS, BR) at 3.0 mL/hL for 4 h at room temperature (25 ± 2 °C), racked, and stored in a cold room at 5 °C (Frilux, RF-054-ESP, Mafra, SC, BR) (Figure 1).

2.2.2. Cryo-Concentration of Apple Must

The cryo-concentration process suggests a discontinuous application process on a small or large scale. A volume of 65 L (12 g/100 mL soluble solids—SS) of depectinized Granny Smith apple must (as obtained in the previous section) was transferred to non-toxic polyethylene buckets (12 L) with lids and frozen (Metalfrio DA 550, SP, BR) at −20 °C for sufficient time to freeze (~24 h). The frozen samples were cut into pieces (approximately 10 cm3 each) and placed in a cheesecloth bag. They were immediately centrifuged (Consul, C2A05ABANA, Rosario, SF, AR) at 20 °C for 5 min at 988× g to force the separation of the solutes from the frozen samples. After centrifugation, the soluble solids of the concentrate samples and the remaining ice were determined using a refractometer (Megabrix, AR1000 S, São Paulo, SP, BR). The same procedure was repeated for the first, second (25 g/100 mL SS), and third (52 g/100 mL SS) steps (Figure 1). In the third step, the residual ice had a low soluble solids content (1.5 g/100 mL SS), and the final volume of cryo-concentrate was 15 L, which was used to standardize the final cider to correct sugar content, aromas, color, acidity, and astringency.
The incorporation of cryo-concentrated Granny Smith juice, due to its elevated acidity, contributes to a more sensorially balanced beverage, eliminating the need for acidity correction with chemical additives.

2.2.3. Hard Cider Processing and Formulation

In this research, a new hard cider was evaluated. Depectinized apple juice from the Gala cultivar, containing 12.2 g/100 mL SS, was placed in two fermenters (50 and 30 L) made of stainless steel AISI 304, with a usable volume of 45 L and 25 L, respectively, temperature control, pressure resistance (9 bar), safety valve, and gasification system (Grupo Musso Inox, Vila Velha, ES, BR). The apple musts were inoculated (106 CFU/mL) with Saccharomyces cerevisiae Reims (Fermol Reims Champagne, PB 2002, AEB Group, BSa, IT). Alcoholic fermentation occurred during 20 days of anaerobiosis at 20 °C. After removing the lees, the two ciders were mixed and homogenized. Sulfite was not evaluated. The dry cider was racked and stored in a cold room at 5 °C (Frilux, RF-054-ESP, Mafra, SC, BR) (Figure 1).
The hard cider was formulated with dry cider and cryo-concentrate apple must. The cryo-concentrate was added until the cider had 45.0 g/L sugar (demi-sec), 0.48 g/100 mL acidity, and 4.5% (v/v) ethanol [13]. The cider was carbonated (2.0 bar), isobarometrically filled into glass bottles (750 mL), capped with non-toxic plastic stoppers, and secured with metal cages (Figure 1).
The hard cider was divided into four groups: “CA” stored at room temperature (18–23 °C); “CR” stored under refrigeration (7 to 8 °C); “P60” pasteurized at 60 °C for 15 min (15 PUs); and “P80” pasteurized at 80 °C for 15 min (20 PUs). Pasteurization began when the temperature at the cold point reached 60 or 80 °C (Figure 1). A bottle was monitored with a thermometer in the stopper.
After heat treatment, the samples were cooled to 25 °C and kept at room temperature (18–23 °C). All samples were monitored after 0, 30, 60, 120, and 180 days. Three bottles were analyzed at each time point. The experiment was carried out in triplicate. The first analysis (time zero) was carried out after a stabilization period (6 h).

2.3. Analysis

The analyses described in this section were carried out on samples of apple must, cryo-concentrate, and hard cider where appropriate.

2.3.1. Microbiological Monitoring

Microbiological analyses for the enumeration of lactic acid bacteria, thermotolerant coliforms, Escherichia coli, and total yeasts/molds, and the detection of the presence or absence of Salmonella spp., were performed according to Silva et al. [14]. Sterilized 0.1% (w/v) peptone saline was used as a diluent for microbial enumeration. Microorganism counts were carried out in triplicates and the average value was expressed in cells per mL.

2.3.2. Quantification of Sugars and Glycerol

The determination of sugar and glycerol content was performed as described by Santos et al. [15] in a high-performance liquid chromatography (HPLC) system (Waters Alliance 2695, Milford, MA, USA) coupled with a refractive index detector (Waters RI 2414, Milford MA, USA) and a Sugar Pak™ column (300 × 6.5 mm i.d.). The areas of the peaks were determined and compared with the standard curves (sucrose, D-glucose, D-fructose, and glycerol). Sugar was expressed as grams per liter of cider.

2.3.3. Physicochemical Analysis

Total titratable acidity was expressed as malic acid (g/L) and volatile acidity as acetic acid (g/L) [16]. The density of the samples was determined using a digital densimeter with an accuracy of 5 × 10−3 kg/m3 (DMA 4500M, Anton Paar, Graz, Austria). The pH was determined using a pH meter (pH 21 m, Hanna, Cotia, BR). The alcohol content was determined with an ebulliometer (010154, Techvision, Guarulhos, SP, BR). Equation (1) was used to calculate PUs [10], where t = time in minutes and T = temperature in degrees Celsius.
PU = t × 1.393(T − 60)

2.3.4. Colorimetric Analysis

The cider colors were expressed in the Commission Internationale de l’Eclairage (CIE) L*, a*, and b* color space coordinates. The samples were analyzed using a digital colorimeter (CM-5-ID, KONICA MINOLTA, Osaka, Japan) with Illuminant D65 and SpectraMagic NX (Color data software CM-S100w, v. 2.6, Osaka, Japan). Colors were obtained by converting CIELAB data (L*, a*, and b*) to other systems such as RGB, HEX (hexadecimal), and CYMK using the NIX® color sensor software (v. 3.0, Osaka, Japan). The chroma parameter (C*) related to color intensity was calculated (2) and the hue angle parameter (h°) was related to tonality (3):
C* = (a2 + b2)1/2
h°= tan−1 (b*/a*) + 180° when a* < 0 and h* = tan−1 (b*/a*) when a* > 0

2.3.5. Phenolic Composition

Total phenolic compounds were determined according to Singleton and Rossi [17]. Absorbance was read at 760 nm (Epoch Microplate Spectrophotometer, Synergy-BIOTEK, Winooski, VT, USA) and concentrations were calculated using a chlorogenic acid curve (TPC = 1612.90 × absorbance; R2 = 0.9932). Results were expressed as milligrams of chlorogenic acid equivalents per liter (mg CEA/L). All analyses were performed in triplicate.
Individual phenolic compounds were analyzed using HPLC (Waters Alliance 2695, Milford, MA, USA), following the method described by Alberti et al. [5]. Before analysis, 10 mL of the samples were frozen at −80 °C (Terroni, Cold 80, São Carlos, SP, BR) and lyophilized (Terroni, LS3000, São Carlos, SP, BR). They were reconstituted with 5 mL of 2.5% acetic acid and methanol solution (3:1 v/v) and then filtered through a 0.22 μm nylon syringe filter (Waters). The system used was an Alliance 2695 with a quaternary pump, degasser, and automatic injector: Waters PDA 2998 photodiode array detectors and a Symmetry C18 column (4.6 × 150 mm, 3.5 μm; Waters) were used at a temperature of 20 °C. The identification and quantification of the compounds were performed by comparison with the retention times and spectra of commercial standards.

2.3.6. In Vitro Antioxidant Capacity

The antioxidant potential was determined using DPPH [18], ABTS [19], and FRAP [20] methods. Absorbances were compared with the standard curves (FRAP = 1111.11 absorbance, R2 = 0.991; DPPH = 3.57 × absorbance, R2 = 0.996 and ABTS = 5.29 × absorbance, R2 = 0.997) of Trolox (10–1000 μmoL/kg). All results are expressed as milligrams of standard per liter of cider.

2.3.7. Statistical Analysis

Data were presented as mean and standard deviation. The homogeneity of variance was tested by Levene’s test or F-test (p ≥ 0.05). Differences between samples were evaluated using Student’s t-test or one-way ANOVA, followed by Fisher’s LSD test. Statistical analysis was performed using Statistica v. 13.3 software (TIBCO Software Inc., Palo Alto, CA, USA).

3. Results and Discussion

The composition of cider, standardized in terms of sugar, ethanol, and acidity through the addition of cryo-concentrate apple must, is shown in Table 1. In a previous work, this beverage demonstrated excellent sensory acceptance, with 81.6% approval and an 85.0% purchase intention rate. One of the main highlights of sensory quality was the intense fruity aroma [13]. The incorporation of cryo-concentrate significantly enhanced the levels of phenolic compounds, resulting in a 57% increase, while antioxidant activity improved by 2.2 to 2.7 times compared to the base cider (Table 1). This increase influenced the color to become more intense and contributed to a more pronounced astringency [5,13]. Functionally, the elevated antioxidant potential added further health benefits (Table 1).
However, this beverage was specifically designed for on-tap consumption, requiring storage at low temperatures (0–2 °C) and exhibiting a limited shelf life of two weeks. Consequently, in this study, different treatment approaches were evaluated, with a special focus on the impact of heat treatment to preserve its quality features as effectively as possible.

3.1. Microbiological Aspects

Contamination with Salmonella spp., Escherichia coli, and thermotolerant coliforms did not occur (<3 NMP/mL) in the samples of hard cider with added cryo-concentrate on any storage day. The selection, washing, and disinfection of fruit and equipment combined with a pH < 4.0 and ethanol of 4.6% (Table 2) ensured the absence of pathogenic microorganisms.
However, molds/yeasts and lactic acid bacteria are difficult to eliminate. These microorganisms are present in high populations on the surface of the fruit, show a certain resistance to hypochlorite treatment [21], and are adapted to the pH and ethanol of ciders. These microorganisms are not pathogenic, but they can cause changes in the composition due to a second alcoholic and malolactic fermentation, which could negatively affect its quality.
Samples stored at room temperature (CA, 18–23 °C) and refrigerated (CR, 7–8 °C) were considered as controls to observe the changes that could occur without heat treatment. CA and CR showed a 40.7- and 22.2-fold increase in yeast/mold populations, respectively, during the first 30 days of storage (Table 2). These increases in both cases significantly altered the chemical composition and were detrimental to the quality of the beverages. Therefore, ciders stored under these conditions exhibited a significantly reduced shelf life, rendering them economically unfeasible. Our pilot-scale experience with this hard cider with added cryo-concentrate, produced without heat treatment and stored between 1 and 3 °C, shows a maximum shelf life of two weeks.
In this study, the heat treatments (P60 and P80) were mild compared to pasteurization to eliminate > 99% of the microorganisms (85 °C for 20 min). These conditions were evaluated to maintain the maximum cider composition. The heat treatment reduced the initial populations of molds and yeasts by 92.9% and 83.3% in the P80 (80 °C for 15 min) and P60 (60 °C for 15 min), respectively (Table 2). This effect depends on the initial population and composition of the cider [22]. In addition, P80 and P60 showed a decrease in yeast population of 4.78 and 2.85 times, respectively, during the first 30 days of storage. This behavior is due to the loss of metabolic functions with the denaturation of proteins in the remaining cells.
The P60 cider can be commercialized at low temperatures (<10 °C) due to the slight change in its physicochemical composition during storage at room temperature (Table 3). Based on microbiological results, this treatment (P60) combined with refrigeration could extend the shelf-life. On the other hand, ciders treated with P80 can be bottled and stored at room temperature (18–23 °C). In this experiment, the P80 ciders reached a shelf life of six months (the maximum period evaluated) due to the low yeast and LAB counts (Table 2).

3.2. Physicochemical Composition

The hard cider CA stored at room temperature (18–23 °C) underwent new alcoholic fermentation, as evidenced by the depletion of residual sugars and a corresponding increase in alcohol content within the first 30 days of storage (Table 3). During this period, the cider, initially classified as demi-sec (45 g/L of residual sugar), transitioned into a dry cider, with residual sugar levels dropping below 9.0 g/L [3].
The CR cider showed the same behavior, but at a slower rate due to the low temperature (7–8 °C). The presence of S. cerevisiae with fermentative viability is evident in the hydrolysis of sucrose (Table 3). These microorganisms release invertase into the medium, which hydrolyses this disaccharide into glucose and fructose [23]. Glycerol plays an important role in the processing of dry cider, providing fullness of flavor [8]. This compound is produced during alcoholic fermentation by S. cerevisiae, which explains its increase in CA samples (Table 2). In CR, its variation was low due to the low temperature. However, P60 and P80 showed a reduction in glycerol during storage. This reduction could be due to the metabolism of lactic acid bacteria, even in low populations (Table 2) [24].
The sugar content was the main parameter that decreased in the P60 samples around 60 days of storage. This indicates a possible slow biotransformation process (Table 3). On the other hand, in the P80 samples only sucrose decreased, and there was even an increase in glucose and fructose, which indicates an acid hydrolysis of sucrose and not by S. cerevisiae invertase. Thus, P80 can still be considered stable after 120 days.
However, in these two treatments (P60 and P80), the color of the samples darkened during storage, whereas this did not occur in the other two treatments (CA and CR) (Table 3 and Figure 2). Hue (tonality) decreased during storage, but the decrease was greater with heat treatment. In CA and CR, the color intensity (chroma) decreased and remained stable, respectively. However, at P60 and P80, the color intensity doubled at the end of 180 days, from straw yellow to a more intense yellow (Table 3, Figure 2B and Figure S1). This darkening of the cider may have occurred slowly via the Maillard reaction due to the incorporation of amino acids and monosaccharides by the cryo-concentrated apple must added to the cider followed by heat treatment.
However, the possibility of residual PPO and POD cannot be excluded. The activities of two groups of enzymes, polyphenol oxidase (PPO) and peroxidase (PDO), are important for the nutritional and sensory properties of apple juices and ciders [25]. PPO mainly catalyzes the hydroxylation of chlorogenic acid, a substrate for enzymatic browning, to ortho-quinones, which are already dark in color and subsequently undergo non-enzymatic secondary reactions by condensation to synthesize the brown pigment melanin [26]. According to Murtaza et al. [27] apple juice enzymes (PPO and POD) retained most of their relative activities after heat treatment at 65–75 °C. This temperature range was comparable to the one employed in this study. Therefore, the possibility of residual PPO and POD activity is high, explaining the enzymatic browning of the cider. The case of CA and CR ciders that did not brown could be explained by the fact that the higher ethanol content (>5.0%) inhibited the action of the enzymes, as ethanol has been studied to inhibit PPO in vegetables [28].
This information is relevant because color is the first factor consumers perceive and influences product attractiveness [29]. Therefore, the heat treatment and ethanol content should be evaluated to avoid or minimize these changes during the prolonged storage of this cider.

3.3. Phenolic Composition and Antioxidant Activity

The initial content of hydroxycinnamic acids was little affected by heat treatment, but cider storage showed more significant changes in the profile of this class (Table 4). Chlorogenic acid (CQA) decreased rapidly in ciders without heat treatment. In P60 ciders, the reduction was visible after 180 days of storage (Table 4). In P80 cider, no decrease in CQA was observed due to the denaturation of the enzymes responsible for this reaction. The reduction in CQA and the increase in caffeic acid can be caused by de-esterification carried out by the hydrolases of S. cerevisiae. This reaction was not observed in P80 ciders, possibly due to the denaturation of these enzymes [2].
Epicatechin decreased during storage in all treatments. On the other hand, catechin increased during storage (Table 4). Similar results were found in French ciders, where epicatechin and catechin compounds decreased and increased, respectively [2]. Dihydrochalcones decreased during storage in the CA and CR treatments but increased in the P60 and P80 heat treatments (Table 4). This means that the presence of enzymes can reduce the levels of these compounds, and their absence can increase them through chemical reactions.
In Table 5, the levels of phenolic compounds (TPC) and antioxidant activity (FRAP and DPPH) at zero storage time were 1.8 to 3.0 times higher than those found in ciders made from dessert apples [5]. The cider without cryo-concentrated apple must had 496 mg/L of total phenols (Table 1).
According to the results, all of the monitored values followed the same trend of decreasing with storage time and with different treatments (Table 5). The heat treatments (P60 and P80) did not affect the content of phenolic compounds at time zero. However, the polyphenol content in all samples decreased by 10% to 17% in 180 days of storage. This reduction was more significant after 60 days of storage (Table 5). The greatest reduction was in the untreated sample (CA). In the other treatments, the reduction was smaller and similar, indicating that the low temperature and pasteurization affected this reaction (10–14%). This suggests that the decrease in phenolic compounds was due to enzymatic and chemical reactions. However, even with these losses, the ciders showed higher TPC values than the initial cider without cryo-concentrate (Table 5).
This reduction in TPC had a direct effect on the antioxidant activity of the ciders. A decrease was observed in all samples, especially after 60 days of storage. In the P60 and P80 samples, losses ranged from 11% to 51%, respectively, after 180 days of storage. The P60 ciders showed a gradual decrease in their antioxidant activities with a FRAP, DPPH, and ABTS of 11.1%, 45.5%, and 50.0%, respectively, after 180 days of storage. In the P80 ciders, these decreases were 32.6%, 43.7% and 51.5%, respectively. The ABTS and DPPH scavenging activities decreased more during storage than the FRAP activity (Table 4). Cider had a greater ability to donate H protons than to reduce free radicals. The decrease in antioxidant activity on pasteurization is also consistent with the loss of vitamin C, which acts as an antioxidant. Odriozola-Serrano et al. [30] suggested that heating significantly affects the loss of ascorbic acid via the aerobic pathway, as ascorbic acid is a heat-sensitive bioactive compound. The data obtained in this study suggest that low-temperature pasteurization (P60) maintained a higher antioxidant content.
In this study, the storage time was the primary factor in hard cider quality. Pasteurizing the cider at 60 °C for 15 min allowed it to be stored for up to 60 days at room temperature (18–23 °C). On the other hand, pasteurization at 80 °C for 15 min made the cider stable in microbiological terms for the maximum time of the study. However, reductions were observed in phenolic compounds, color, and antioxidant activity, due to chemical reactions. Even so, the values were still higher than those found in ciders processed with table apples without the addition of cryo-concentrate. However, further studies need to be carried out on the sensory impact of heat treatments on this cider to minimize quality losses and offer a product with an adequate shelf life.

4. Conclusions

The effect of heat treatment on hard cider enriched with cryo-concentrated apple must was evaluated during 180 days of storage. The control hard ciders (CA and CR) provided insights into the biochemical and chemical changes during storage.
The counts of microorganisms remaining after heat treatment (P60 and P80) gradually decreased during storage. The sugars in P60 samples were the primary indicators of cider instability, particularly around 60 days of storage.
During storage, color intensity doubled, while phenolic content and antioxidant activity declined. Pasteurization at 60 °C and 80 °C extended shelf life to ~2 and 6 months, respectively, with P80 being the most effective for cider with cryo-concentrate stored at room temperature. This new hard cider formulation, made entirely from dessert apples, has the potential to be an innovative product, with heat treatment that guarantees a long shelf life and quality.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/fermentation11040188/s1, Figure S1: Photo of hard ciders with different treatments and storage periods.

Author Contributions

Formal analysis, M.d.M.C. and I.M.M.S.S.; writing—original draft preparation, M.d.M.C. and I.M.M.S.S.; writing—review and editing, R.D.M.d.S., I.M.D., A.A. and A.N.; methodology, I.M.D. and A.A.; supervision, A.N.; data curation and statistic, R.D.M.d.S.; resources, A.N.; project management, A.N.; fundraising, A.N. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the National Council for Scientific and Technological Development (CNPq) [grant No. 302797/2023-8; project No. 406799/2023-7], the Araucária Foundation (FA), and the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior -Brasil (CAPES) [grant finance code 001].

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The raw data supporting the conclusions of this article will be made available by the authors on request.

Acknowledgments

The authors express their gratitude to the funding institutions and acknowledge the analytical infrastructure provided by the Apple Working Group (GTM) and the Multi-User Laboratory Complex (C-Labmu) at the State University of Ponta Grossa.

Conflicts of Interest

The authors declare no conflicts of interest.

References

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Figure 1. Flowchart of cider processing with the addition of cryo-concentrate and preservation treatments.
Figure 1. Flowchart of cider processing with the addition of cryo-concentrate and preservation treatments.
Fermentation 11 00188 g001
Figure 2. Cartesian position of the samples in the CIELAB* system (A) and conversion of the CIELAB system into colors (B). Note: L*: luminosity; a*: green–red parameter; b*: blue-yellow parameter. The number shown in the figure = sample code—day of storage (i.e., 1: CA-0); 1: CA-0; 2: CR-0; 3: P80-0; 4: P60-0; 5: CA-30; 6: CR-30; 7: P80-30; 8: P60-30; 9: CA-60; 10: CR-60; 11: P80-60; 12: P60-60; 13: CA-180; 14: CR-180; 15: P80-180; and 16: P60-180. CA: ambient control (18–23 °C); CR: cider stored under refrigeration (7–8 °C); P80: cider pasteurized at 80 °C for 15 min; and P60: cider pasteurized at 60 °C for 15 min. Data from ciders stored for 120 days were not used.
Figure 2. Cartesian position of the samples in the CIELAB* system (A) and conversion of the CIELAB system into colors (B). Note: L*: luminosity; a*: green–red parameter; b*: blue-yellow parameter. The number shown in the figure = sample code—day of storage (i.e., 1: CA-0); 1: CA-0; 2: CR-0; 3: P80-0; 4: P60-0; 5: CA-30; 6: CR-30; 7: P80-30; 8: P60-30; 9: CA-60; 10: CR-60; 11: P80-60; 12: P60-60; 13: CA-180; 14: CR-180; 15: P80-180; and 16: P60-180. CA: ambient control (18–23 °C); CR: cider stored under refrigeration (7–8 °C); P80: cider pasteurized at 80 °C for 15 min; and P60: cider pasteurized at 60 °C for 15 min. Data from ciders stored for 120 days were not used.
Fermentation 11 00188 g002
Table 1. Physicochemical composition of dry cider (Gala), cryo-concentrate apple must (Granny Smith), and hard cider with added cryo-concentrate.
Table 1. Physicochemical composition of dry cider (Gala), cryo-concentrate apple must (Granny Smith), and hard cider with added cryo-concentrate.
Analytical ParametersDry CiderCryo-Concentrate
Apple Must
Hard Cider with
Cryo-Concentrate
Total sugar (g/100 mL)0.56 ± 0.0152.2 ± 0.34.48 ± 0.01
Sucrose (g/100 mL)<LOD8.3 ± 0.10.17 ± 0.01
Glucose (g/100 mL)<LOD13.2 ± 0.11.39 ± 0.01
Fructose (g/100 mL)0.56 ± 0.0130.6 ± 0.12.91 ± 0.01
Glycerol (g/100 mL)0.38 ± 0.01<LOD0.29 ± 0.01
Ethanol (% v/v)6.75 ± 0.07 - 4.6 ± 0.1
Density (g/cm)0.993191.214581.01446
pH3.79 ± 0.013.23 ± 0.023.55 ± 0.01
Total acidity (g MA/L)3.51 ± 0.0128.11 ± 0.024.81 ± 0.05
Volatile acidity (g AC/L)1.0 ± 0.20.6 ± 0.20.9 ± 0.1
TFC (mg CAE/L)496 ± 85440 ± 80870 ± 26
FRAP (µmol TE/L)734 ± 611,996 ± 2141911 ± 30
DPPH (µmol TE/L)546 ± 444755 ± 851217 ± 17
ABTS (µmol TE/L)1061 ± 6013,024 ± 972923 ± 134
L*91.07 ± 0.0180.90 ± 0.0394.0 ± 0.3
a*−0.71 ± 0.0112.62 ± 0.02−1.13 ± 0.03
b*39.30 ± 0.1074.60 ± 0.0930.6 ± 0.1
°Hue88.70 ± 0.0180.40 ± 0.0392.11 ± 0.05
Chroma39.30 ± 0.1075.66 ± 0.0830.6 ± 0.1
Note: LOD: limit of detection; TFC: total phenolic compounds; MA: malic acid; AC: acetic acid; CAE: chlorogenic acid equivalent; TE: Trolox equivalent; L*: lightness; a*: green/red color parameter; and b*: blue/yellow color parameter.
Table 2. The average population of molds/yeasts and lactic acid bacteria in hard ciders with added cryo-concentrate, with different heat treatments, during storage.
Table 2. The average population of molds/yeasts and lactic acid bacteria in hard ciders with added cryo-concentrate, with different heat treatments, during storage.
Sample Storage DaysMolds/Yeasts
(Cells/mL)
Lactic Acid Bacteria
(Cells/mL)
CA02.7 × 1042.5 × 105
301.1 × 106<1.0 × 102
601.0 × 1051.9 × 105
1204.7 × 1048.4 × 105
1804.5 × 1038.9 × 106
CR02.7 × 1042.5 × 105
306.0 × 1054.0 × 105
605.0 × 1055.7 × 104
1202.1 × 1054.2 × 104
1802.2 × 1032.7 × 105
P6003.7 × 103presence (<4.0 × 101)
301.3 × 103<1 × 102
601.8 × 103presence (<4.0 × 102)
1201.1 × 103presence (<4.0 × 102)
1805.2 × 102presence (<4.0 × 102)
P8001.9 × 103presence (<4.0 × 101)
30presence (<4.0 × 102)<1 × 102
601.6 × 103presence (<4.0 × 102)
1209.5 × 102presence (<4.0 × 102)
1803.3 × 101presence (<4.0 × 102)
Note: CA: ambient control (18–23 °C); CR: cider stored under refrigeration (7–8 °C); P60: cider pasteurized at 60 °C for 15 min; and P80: cider pasteurized at 80 °C for 15 min.
Table 3. Monitoring of the physicochemical composition (g/100 mL) of ciders, with different heat treatments, during storage.
Table 3. Monitoring of the physicochemical composition (g/100 mL) of ciders, with different heat treatments, during storage.
SampleStorage
Day
Total SugarSucroseGlucoseFRUGLYDensity
(kg/m3)
Ethanol
(% v/v)
pHTotal
Acidity
Volatile
Acidity
°HueChroma
CA04.68 a ± 0.010.60 a ± 0.011.39 a ± 0.012.91 a ± 0.010.29 i ± 0.011014.5 d4.6 fg ± 0.13.55 d ± 0.010.48 b ± 0.050.08 c ± 0.0192.1 a ± 0.130.6 e ± 0.1
30<LOD<LOD<LOD<LOD0.44 b ± 0.01993.5 h7.4 b ± 0.13.98 ab ± 0.040.31 c ± 0.010.20 a ± 0.0187.9 bc ± 0.124.4 h ± 0.2
60<LOD<LOD<LOD<LOD0.43 c ± 0.01993.8 h7.1 c ± 0.24.01 a ± 0.030.3 c ± 0.10.08 c ± 0.0088.7 b ± 0.123.8 hi ± 0.1
120<LOD<LOD<LOD<LOD0.44 b ± 0.01993.7 h7.4 b ± 0.23.98 ab ± 0.010.3 c ± 0.10.09 c ± 0.0087.9 bc ± 0.124.1 h ± 0.2
180<LOD<LOD<LOD<LOD0.45 a ± 0.01993.7 h7.7 a ± 0.13.95 b ± 0.020.3 c ± 0.10.11 b ± 0.0287.2 c ± 0.024.3 h ± 0.1
CR04.68 a ± 0.010.60 a ± 0.011.39 a ± 0.012.91 a ± 0.010.29 i ± 0.011014.5 d4.6 fg ± 0.13.55 d ± 0.010.48 b ± 0.050.08 c ± 0.0192.1 a ± 0.130.6 e ± 0.2
304.08 b ± 0.03<LOD1.27 b ± 0.012.81 b ± 0.020.31 g ± 0.011013.8 d4.9 f ± 0.13.63 c ± 0.010.49 b ± 0.050.06 cd ± 0.0190.7 ab ± 0.133.4 d ± 0.1
603.86 c ± 0.01<LOD1.19 c ± 0.012.66 c ± 0.010.33 e ± 0.011012.0 e5.0 f ± 0.13.67 c ± 0.020.48 b ± 0.030.08 c ± 0.0090.2 ab ± 0.133.7 d ± 0.2
1203.53 cd ± 0.01<LOD1.06 c ± 0.012.59 d ± 0.030.32 ef ± 0.011011.6 f5.9 e ± 0.13.65 c ± 0.020.49 b ± 0.020.08 c ± 0.0289.7 b ± 0.331.2 e ± 0.2
1803.46 d ± 0.03<LOD0.93 d ± 0.012.53 d ± 0.030.31 g ± 0.011011.2 g6.7 d ± 0.13.63 c ± 0.030.5 ab ± 0.10.06 cd ± 0.0189.3 b ± 0.328.7 f ± 0.6
P6004.65 a ± 0.030.60 a ± 0.011.26 b ± 0.012.74 bc ± 0.030.35 d ± 0.011014.5 d4.7 ef ± 0.13.55 d ± 0.020.49 b ± 0.050.12 b ± 0.0292.1 a ± 0.222.5 i ± 0.1
304.34 b ± 0.030.54 ab ± 0.011.15 c ± 0.022.64 c ± 0.010.30 h ± 0.011014.8 c4.50 f ± 0.013.63 c ± 0.010.48 b ± 0.070.07 c ± 0.0187.6 c ± 0.130.5 e ± 0.2
604.41 ab ± 0.040.40 bc ± 0.011.24 b ± 0.012.69 c ± 0.040.30 h ± 0.011014.8 c4.7 ef ± 0.13.64 c ± 0.010.48 b ± 0.070.06 cd ± 0.0185.3 d ± 0.238.3 b ± 0.4
1204.06 b ± 0.010.30 d ± 0.011.16 c ± 0.022.50 d ± 0.010.26 j ± 0.011014.9 b4.50 f ± 0.023.63 c ± 0.010.48 b ± 0.040.05 d ± 0.0182.5 e ± 0.140.2 b ± 0.4
1803.71 c ± 0.040.21 e ± 0.011.08 c ± 0.012.32 e ± 0.030.23 k ± 0.011015.1 b4.7 ef ± 0.13.62 c ± 0.010.5 ab ± 0.10.05 d ± 0.0179.7 e ± 0.244.4 a ± 0.4
P8004.63 a ± 0.010.60 a ± 0.011.17 c ± 0.012.71 bc ± 0.010.31 g ± 0.011015.0 b4.6 fg ± 0.13.56 d ± 0.020.49 b ± 0.060.10 bc ± 0.0190.5 ab ± 0.225.3 g ± 0.4
304.41 ab ± 0.030.55 ab ± 0.011.23 b ± 0.012.65 c ± 0.030.31 g ± 0.011015.3 a4.6 fg ± 0.13.63 c ± 0.010.48 b ± 0.020.06 cd ± 0.0287.1 c ± 0.131.2 e ± 0.1
604.48 a ± 0.020.48 b ± 0.011.28 b ± 0.012.80 b ± 0.020.30 h ± 0.011015.0 b4.6 fg ± 0.23.64 c ± 0.010.48 b ± 0.030.10 bc ± 0.0184.6 d ± 0.137.9 c ± 0.2
1204.34 b ± 0.020.40 bc ± 0.011.26 b ± 0.012.50 d ± 0.010.28 i ± 0.011015.0 b4.6 fg ± 0.23.61 c ± 0.010.5 ab ± 0.10.08 c ± 0.0181.9 e ± 0.142.8 ab ± 0.2
1804.21 b ± 0.020.31 d ± 0.011.25 b ± 0.012.76 bc ± 0.030.25 j ± 0.011015.0 b4.6 fg ± 0.13.58 cd ± 0.020.51 a ± 0.050.06 cd ± 0.0179.3 f ± 0.144.4 a ± 0.2
Note: CA: ambient control (18–23 °C); CR: cider stored with refrigeration (7–8 °C); P80: cider pasteurized at 80 °C for 15 min; P60: cider pasteurized at 60 °C for 15 min; FRU: fructose; GLY: glycerol; LOD: limit of detection; different lowercase letters in the same column indicate a significant difference (p < 0.05) according to Fisher’s LSD test.
Table 4. Evolution of individual phenolic compounds (mg/L) in hard ciders with added cryo-concentrate, with different heat treatments, during storage.
Table 4. Evolution of individual phenolic compounds (mg/L) in hard ciders with added cryo-concentrate, with different heat treatments, during storage.
SamplesStorage DaysHydroxycinnamic AcidFlavan-3-OisDihydrochalcones
CQACAACMACATEPCPB2PLZF2X
CA011.18 c ± 0.0613.4 d ± 0.23.27 f ± 0.0410.4 cd ± 0.316.7 c ±0.57.8 e ± 0.27.29 c ± 0.039.3 b ± 0.4
303.77 f ± 0.0215.07 c ± 0.032.95 g ± 0.0211.47 b ± 0.0416.1 d ± 0.99.00 c ± 0.076.46 e ± 0.057.94 d ± 0.03
601.30 h ± 0.0116.96 b ± 0.043.59 d ± 0.0111.24 bc ± 0.0214.11 g ± 0.018.84 c ± 0.045.28 g ± 0.017.64 e ± 0.03
1200.71 i ± 0.0118.85 ab ± 0.033.83 d ± 0.0112.18 ab ± 0.049.45 k± 0.039.97 c ± 0.053.3 h ± 0.15.89 f ± 0.01
1800.12 j ± 0.0420.74 a ± 0.044.07 c ± 0.0313.13 a ± 0.049.1 k± 0.711.1 b ± 0.11.32 i ± 0.044.14 g ± 0.05
CR011.2 c ± 0.113.4 d ± 0.23.27 f ± 0.0410.4 cd ± 0.316.72 c ±0.507.8 e ± 0.27.29 c ± 0.039.3 b ± 0.4
306.41 e ± 0.019.91 g ± 0.032.45 h ± 0.017.06 f ± 0.0810.38 j ± 0.086.1 f ± 0.45.22 g ± 0.026.09 f ± 0.01
609.06 d ± 0.0115.06 c ± 0.043.87 d ± 0.0610.2 d ± 0.114.55 fg ± 0.048.1 d ± 0.17.4 c ± 0.18.7 c ± 0.2
1206.32 e ± 0.0116.7 b ± 0.34.28 c ± 0.039.45 e ± 0.0211.55 ij ± 0.018.09 d ± 0.077.15 c ± 0.037.60 e ± 0.03
1803.58 f ± 0.0416.94 b ± 0.054.69 c ± 0.058.7 e ± 0.28.46 l ± 0.068.1 d ± 0.16.9 d ± 0.16.5 f± 0.3
P60011.7 b ± 0.411.9 e ± 0.42.9 g ± 0.110.30 d ± 0.0317.26 b ± 0.508.4 c ± 0.26.79 e ± 0.028.49 c ± 0.05
3010.68 c ± 0.0214.80 cd ± 0.044.15 c ± 0.0311.1 b ± 0.118.52 a ± 0.0313.3 a ± 0.27.86 c ± 0.029.14 b ± 0.03
608.51 d ± 0.0114.68 cd ± 0.064.33 c ± 0.0310.65 c ± 0.0114.9 f ± 0.36.0 f ± 0.27.3 c ± 0.19.2 b ± 0.1
1205.41 e ± 0.0117.71 b ± 0.055.86 b ± 0.0311.1 b ± 0.112.4 i ± 0.54.8 g ± 0.17.95 c ± 0.019.9 a ± 0.1
1802.32 g ± 0.0320.74 a ± 0.057.39 a ± 0.0711.55 b ± 0.0110.9 j ± 0.33.1 h ± 0.28.6 b ± 0.110.6 a ± 0.1
P80010.5 c ± 0.310.2 g ± 0.32.30 h ± 0.048.35 e ± 0.315.5 e ± 0.68.9 c ± 0.25.8 f ± 0.36.41 f ± 0.05
3013.0 a ± 0.211.2 ef ± 0.12.92 g ± 0.0210.1 d ± 0.115.87 de ± 0.038.3 cd ± 0.36.60 e ± 0.038.71 c ± 0.02
6012.0 b ± 0.212.0 e ± 0.13.47 d ± 0.019.4 e ± 0.213.4 h ± 0.15.07 g ± 0.067.11 d ± 0.18.39 c ± 0.01
12012.2 a ± 0.213.8 d ± 0.34.64 c ± 0.0510.65 c ± 0.0111.55 ij ± 0.031.98 i ± 0.028.44 b ± 0.019.1 b ± 0.2
18012.5 a ± 0.215.7 c ± 0.25.82 b ± 0.0311.9 b ± 0.29.71 k ± 0.03<LOQ9.77 a ± 0.039.81 b ± 0.04
Note: CA: ambient control (18–23 °C); CR: cider stored with refrigeration (7–8 °C); P80: cider pasteurized at 80 °C for 15 min; and P60: cider pasteurized at 60 °C for 15 min. CQA: chlorogenic acid; CAA: caffeic acid; CMA: p-coumaric acid; CAT: (+)-catechin; EPC: (−)-epicatechin; PB2: procyanidin B2; PLZ: phloridzin; F2X: phloretin-2-xiloglucoside; and <LOQ: less than the limit of detection. Different small letters in the same column indicate a significant difference (p < 0.05) according to Fisher’s LSD test.
Table 5. Evolution of total phenolic compounds and antioxidant activities of hard ciders with added cryo-concentrate, with different treatments, during storage.
Table 5. Evolution of total phenolic compounds and antioxidant activities of hard ciders with added cryo-concentrate, with different treatments, during storage.
SampleStorage
Days
TPC
(mg CAE/L)
DPPH
(µmol TE/L)
ABTS
(µmol TE/L)
FRAP
(µmol TE/L)
CA0870 ab ± 261217 a ± 172923 b ± 1341911 b ± 30
30780 ef ± 261102 b ± 303076 a ± 231862 bc ± 53
60810 cde ± 401123 b ± 172900 b ± 651900 bc ± 161
120768 efg ± 27839 e ± 422345 d ± 331564 fg ± 23
180719 gh ± 26556 h ± 71790 h ± 741229 i ± 18
CR0870 ab ± 261217 a ± 172923 b ± 1341911 b ± 31
30845 abc ± 52938 d ± 1012959 ab ± 932075 a ± 45
60830 cd ± 11967 cd ± 332946 ab ± 2671819 bc ± 175
120806 cde ± 25754 f ± 282387 d ± 451670 e ± 57
180783 ef ± 12542 h ± 181828 g ± 121506 g ± 81
P600875 a ± 151140 b ± 1053011 ab ± 691913 b ± 73
30760 efg ± 311129 b ± 222870 e ± 341764 cde ± 112
60770 efg ± 9956 cd ± 412154 e ± 191580 fg ± 115
120777 ef ± 36788 f ± 281846 g ± 221627 f ± 85
180788 ef ± 46621 g ± 531504 i ± 221700 de ± 33
P800860 b ± 101012 c ± 262910 b ± 491819 bcd ± 41
30813 cde ± 151118 b ± 552887 bc ± 711588 fg ± 15
60795 def ± 541156 ab ± 332678 d ± 1441554 fg ± 7
120769 efg ± 27862 e ± 322044 f ± 441389 h ± 15
180743 fg ± 9569 h ± 431410 j ± 101225 i ± 40
Note: CA: ambient control (18–23 °C); CR: cider stored under refrigeration (7–8 °C); P80: cider pasteurized at 80 °C for 15 min; and P60: cider pasteurized at 60 °C for 15 min. TPC: total phenolics compounds. The results are expressed as mean ± standard deviation. Different small letters in the same column indicate a significant difference (p < 0.05) according to Fisher’s LSD test.
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Carraro, M.d.M.; Sola, I.M.M.S.; Santos, R.D.M.d.; Demiate, I.M.; Alberti, A.; Nogueira, A. Impact of Heat Treatment on Hard Cider Enriched with Cryo-Concentrated Apple Must: Microbiological Profile, Functional Properties, and Storage Stability. Fermentation 2025, 11, 188. https://doi.org/10.3390/fermentation11040188

AMA Style

Carraro MdM, Sola IMMS, Santos RDMd, Demiate IM, Alberti A, Nogueira A. Impact of Heat Treatment on Hard Cider Enriched with Cryo-Concentrated Apple Must: Microbiological Profile, Functional Properties, and Storage Stability. Fermentation. 2025; 11(4):188. https://doi.org/10.3390/fermentation11040188

Chicago/Turabian Style

Carraro, Matheus de Melo, Isabela Maria Macedo Simon Sola, Raul Dias Moreira dos Santos, Ivo Mottin Demiate, Aline Alberti, and Alessandro Nogueira. 2025. "Impact of Heat Treatment on Hard Cider Enriched with Cryo-Concentrated Apple Must: Microbiological Profile, Functional Properties, and Storage Stability" Fermentation 11, no. 4: 188. https://doi.org/10.3390/fermentation11040188

APA Style

Carraro, M. d. M., Sola, I. M. M. S., Santos, R. D. M. d., Demiate, I. M., Alberti, A., & Nogueira, A. (2025). Impact of Heat Treatment on Hard Cider Enriched with Cryo-Concentrated Apple Must: Microbiological Profile, Functional Properties, and Storage Stability. Fermentation, 11(4), 188. https://doi.org/10.3390/fermentation11040188

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