The Study of Combination of Biodegradable Packaging and Biocoating with Lactic Acid Bacteria as a Green Alternative for Traditional Packaging in Gouda Cheese

Abstract

Biodegradable packaging, both alone and in combination with acid whey protein coatings, has been used to pack fresh Gouda cheese to improve preservation prior ripening or storage. This study involved three key components: (i) the selection of biodegradable packaging (BP), (ii) the development of a plain liquid acid whey protein concentrate, pectin-based edible coating (BP + Ch + Coating), and (iii) the incorporation of at least 6 log₁₀ CFU (colony forming units) mL⁻¹ Lacticaseibacillus paracasei (BP + Ch + Coating + Lp) and Lactobacillus helveticus (BP + Ch + Coating + Lh) strains. The created compositions were compared with cheese packed in conventional polyethylene (PE) packaging to evaluate their overall synergy effect in reducing microbiological spoilage and influencing chemical parameters in Gouda cheese during 45 days of ripening and cold storage. The evaluation included microbiological analysis (total LAB, Enterobacteriaceae spp., and fungi CFU) and quality assessment of pH, moisture content, water activity, texture, and colour (CEI system) during ripening and shelf life. Although biodegradable packaging (BP) alone did not protect the cheese effectively compared to conventional packaging (EVA/PE/EPC/PVDC), the combination of biodegradable packaging with a coating (BP + Ch + Coating) showed protective properties against Enterobacteriaceae spp. and mould, maintaining moisture, pH, and colour during ripening and storage. Incorporation of L. helveticus (BP + Ch + Coating + Lh) into the coating efficiently decreased the growth of fungi.

1. Introduction

Gouda cheese is listed among the most popular cheeses in the world. Due to its chemical attributes—high fat and protein concentration—this semi-hard cheese can be highly susceptible to microbial hazards growing on the surface during ripening and storage that may change its quality [1]. Microorganisms that spoil dairy products vary greatly due to the potential effects of initial preparation, formulation, processing, packaging, storage, and handling conditions.

Currently, Gouda cheese is packed in conventional multilayer shrinkable Ethylene VinylAcetate/Polyethylene/Ethylene Propylene Copolymer/PolyVinylidene Chloride (EVA/PE/EPC/PVDC), which is a widely used plastic material known for its durability and versatility. EVA/PE/EPC/PVDC is characterized by excellent barrier properties and its ability to protect products and reduce water evaporation during ripening or storage. However, EVA/PE/EPC/PVDC is a single-use packaging ending up as non-degradable waste that persists in the environment, contributing to plastic pollution [2]. The concerns of negative environmental impact caused by using non-biodegradable plastic-based packaging materials force the disposable packaging industry to rethink packaging. Thus, comprehensive research has been conducted to develop and apply bio-based polymers made from a variety of agricultural commodities and/or food waste products [3,4]. These biopolymers—polylactic acid (PLA), polyhydroxyalkanoates (PHA), and starch-based polymers—are considered “green” due to their degradability [5], i.e., their ability to break down into water and carbon dioxide through microbial activity [6,7], with microbes using the resulting oligomers as carbon sources [8]. This increased interest in the use of bio-based materials aimed at improving biodegradation and eco-design based on the incorporation of recycled (post-industrial and post-consumption) and recyclable plastic materials was intensified after EU legislation on Packaging and Packaging Waste was updated [9].

However, products packed into biodegradable packaging materials often have a shorter shelf life compared to traditional or conventional packaging materials. They may be more susceptible to moisture, heat, and other environmental conditions, which can affect their structural integrity and performance over time [10]. Due to their susceptibility to the above-mentioned conditions, their applicability and effectiveness in cheese protection during ripening and storage require comprehensive studies.

On the other hand, there are coatings and films constituting thin layers of edible materials that have been successfully tested to substitute or complement synthetic packaging [11] and prolong the shelf life of cheese regarding quality and safety [12]. According to Karaman et al. (2015) [13], they are not intended to replace conventional packaging materials but to provide an additional layer for cheese preservation that may aid in reducing the costs of the cheese by reducing the amount of traditional packaging necessary.

Whey, a major by-product of the dairy industry, is widely used as a biodegradable material that, after heat-denaturation of whey proteins, produces transparent and flexible films with excellent water vapour, gas, and oil barrier properties [14]. Employing acid whey protein concentrate (LAWPC) in its liquid form not only provides a good base for the coating formulation but also reintroduces LAWPC back into food production, bringing along environmental and economic benefits as well [15]. The efficiency of an edible coating in preserving food quality is mainly related to its moisture and gas barrier properties. However, the incorporation of antimicrobial compounds into coatings could aid in improving the quality and shelf life of food products [16]. Numerous natural antimicrobial substances have been tested and isolated from local sources, for instance, indigenous lactic acid bacteria (LAB) [17]. Lactobacilli are able to produce antimicrobial compounds that can inhibit the growth of undesirable microorganisms or reduce microorganism load [18,19]. By incorporating lactobacilli into coating, it may be possible to create a protective barrier that helps to prevent the growth of surface microflora. It has been reported that application of protective cultures requires a proper carrier to provide protection and support the survival of the strain on the cheese matrix [17,20]. As it has been reported in our previous studies, LAWPC–pectin-based edible coating is an effective vehicle for living cells able to facilitate survival while retaining cheese freshness during cold storage [21,22]. To our knowledge, the applicability of such biocoating for protection of cheese during ripening and storage has not been investigated.

Thus, this work aimed to study the biopackaging (Bp) effectiveness by combining it with biocoating and incorporating indigenous antimicrobial L. paracasei and L. helveticus strains in it.

2. Materials and Methods

2.1. Materials

Six commercial biodegradable packaging materials were tested: polyolefin shrink film (PTC); BOPP Propafilm P2GAF and PropaFresh (Innovia, Wigton, UK), CERAMIS (PLA with SiO₂ coating) (Amcor, Zürich, Switzerland); biodegradable cheese storage sheets (derived from wood-based cellulose fibres; Formaticum, San Francisco, CA, USA); and PLA with Nature Flex coating (Mixpack, Tallinn, Estonia).

Liquid acid whey protein concentrate (LAWPC) was supplied from dairy plant AB Kauno pienas, Kaunas, Lithuania. LAWPC was produced by ultrafiltration using fresh acid whey. The LAWPC (12.34% dry matter, 2.24% protein, 1.77% fat, and 5.55% other solids) was frozen at −18 °C until use. Before coating preparation, the LAWPC was thawed at 4 °C for 24 h.

Glycerol (99% purity) as a plasticizer, apple pectin as a thickener, surfactant Tween 80 (Quality 200), and sunflower oil were supplied by Sigma-Aldrich (St. Louis, MO, USA).

Lactobacillus helveticus MI-LH3 (L. helveticus) and Lacticaseibacillus paracasei A11 (L. paracasei) strains, previously isolated from fermented bovine milk (37 °C 96 h), were chosen as bioprotective strains for coating preparation and stored at −80 °C in MRS broth (Oxoid, Basingstoke, UK) in the presence of 30% glycerol. These indigenous strains were carefully selected for their antifungal and probiotic properties in our previous studies [21,22]. Before experiments, strains were revitalized in MRS broth by growing for 48 h at 37 °C.

Fresh Gouda cheese was supplied from a local cheese factory (Smiltenes piens, Smiltene, Latvia).

2.2. Biocoating

2.2.1. Biopackaging

The biodegradable packaging materials were tested and selected as promising materials for study. The growth of mould and yeast on the surface of the cheese at various stages of ripening and storage was visually evaluated. Surface growth evaluation was noted as follows: no visible growth (−) or a thin layer or bloom on the cheese’s surface (+). The visual evaluation results revealed that cheese packed in DECOLINE^® DCL-BD material did not show any visible growth of fungi; therefore, all experiments in the present study were performed with this material (coded Bp6).

The packaging material technical data are summarized in

Table 1. The characteristics of materials used in the study.

Sample No.	Material	Thickness, µm	Water Vapor Transmission Rate g/m2 24 h (ASTM F 1249-20; 38 °C, 90% RH)	Visual Evaluation of Fungi Growth
1	PropaFresh, Innovia	35 ± 2	3.89	+
2	BOPP Propafilm P2GAF, Innovia	35 ± 2	15.18	+
3	CERAMIS PLA with SiO2 coating, Amcor	45 ± 2	32.67	+
4	Biodegradable cheese storage sheets (derived from wood-based cellulose fibres), Formaticum	25 ± 2	437.47	+
5	PLA with Nature Flex coating, Mixpack	35 ± 2	35.50	+
6	DECOLINE® DCL-BD—biodegradable polyolefin multi-layer shrink film, Dekofilm Polska, Emilianów, Poland (shrinkage 93 °C is MD 22/TD 22)	20 ± 1	29.20	−
7	Conventional packaging BK3550K, Sealed Air, EVA/PE/EPC/PVDC (Ethylene VinylAcetate/Polyethylene/Ethylene Propylene Copolymer/PolyVinylideneChloride)	52 ± 2	17.00	+

.

2.2.2. Coating Solutions

All coating formulations were prepared as described by Vasiliauskaite et al. (2023) [22] with slight modifications. The blank coating formulation was prepared in ambient conditions by adding predetermined amounts of ingredients (5% glycerol (w/w), 2% pectin (w/w), 0.2% Tween 80 (w/w), and 2% sunflower oil (w/w)) in AWPC followed by homogenization and, subsequently, pasteurization of coating media. L. helveticus/L. paracasei (0.2 g/100 g, respectively) after cultivation in AWPC for 48 h was added to the coating (coded as Coating + Lp, Coating + Lh) and thoroughly mixed in.

2.2.3. Experimental Design

The unripened Gouda cheese blocks were cut in a sterile constant airflow box into uniform pieces with an average weight of 110 ± 5 g.

A 2:5 factorial experiment design (2 different packaging sets × 5 time points) was implemented during this study (Figure 1). Accordingly, four sets of experimental cheese samples were prepared, namely Bp6 combined with plain coating (Bp6 + Ch + Coating), Bp6 combined with coating with incorporated L. paracasei (Bp6 + Ch + Coating + Lp), coating with incorporated L. helveticus (Bp6 + Ch + Coating + Lh), and blank cheese in biopackaging (Bp6 + Ch). A reference set of blank cheese in conventional packaging (Pp + Ch) was prepared next to biopackaging sets.

The pre-cut cheese pieces were immersed into coating solution, dried for one hour, then packed into Bp6 or Pp vacuum bags. Each Bp6 and Pp bag was manually prepared by heat sealing (165 °C, 700 Pa, 7 s) on the HST-H6 heat seal tester PARAM (Labthink, Jinan, China) along the long and then short edges. After cheese insertion in the bags, the Bp6 and Pp bags were vacuum sealed using a vacuum sealer (Status d.o.o., Metlika, Slovenia).

The combination of coating and biopackaging (biocoat) was made with the purpose of studying the ultimate mutual/synergistic impact of the biocoat. The sample sets were stored in a ripening chamber at 12 °C and φ = 80% (φ—relative humidity) for 45 days and, after that, kept refrigerated at 4 °C for an additional 160 days. The samples were examined on days 1, 15, 30, 45, and 205. The full names of the samples and their abbreviations are presented in Figure 1.

The experiment was duplicated under identical conditions, with 6 cheese pieces allocated to each treatment.

2.3. Chemical and Physical Analysis

2.3.1. Moisture Content and Weight Loss

The moisture analyser XM 120 HE 53 (Precisa GRavimetrics AG, Dietikon, Switzerland) was used to measure the moisture content of the cheese.

Cheese weight loss was determined by calculating the weights of samples prior to ripening and during ripening. The verified balance KERN (Frankfurt am Main, Germany) with precision ±0.001 g was used for sample weighing. Loss (WL) was calculated using Equation (1):

WL (%) = (m₁ − m₂)/m₁ × 100,

(1)

where

• m₁—initial cheese weight, g;
• m₂—cheese weight during ripening, g.

2.3.2. pH Value

The pH was measured at room temperature using a benchtop Mettler Toledo JENWAY 3510 pH-meter (Barloworld Scientific Ltd., Essex, UK) after calibration. The 10 g of cheese in 30 mL of distilled water was homogenized (Lawson Scientific, Ningbo, China) and analyzed directly.

2.3.3. Water Activity

Water activity (a_w) was measured using a LabSwift-a_w water activity meter (LabSwift-aw, Novasina AG, Lachen, Switzerland) by grinding cheese and immediately placing it onto a measuring plate for analysis.

2.3.4. Colour Change

The colour of samples was measured in a CIE L*a*b* colour system using a Tristimulus Colourimeter, measuring colour parameters by Colour Tec PCM/PSM. Colour values were recorded as L* (brightness), a* (−a, greenness, +a, redness), and b* (−b, blueness, +b, yellowness) [23]. The measurements were repeated 5 times on different randomly selected locations on the surface of each sample. For evaluation of colour change, the total colour difference (∆E*) was calculated between measurements before packaging cheese samples and during the storage time according to Equation (2). Sample colour was assessed as the total colour difference (ΔE*); it was calculated according to the following equation:

$$\Delta {\mathrm{E}}^{*}=\sqrt{{({\mathrm{L}}^{*}-{\mathrm{L}}_0^{*})}^2+{({\mathrm{a}}^{*}-{\mathrm{a}}_0^{*})}^2+{({\mathrm{b}}^{*}-{\mathrm{b}}_0^{*})}^2 },$$

(2)

where

• ${\mathrm{L}}^{*},{\mathrm{a}}^{*},{\mathrm{b}}^{*}$—value of sample colour components measured before packaging;
• ${\mathrm{L}}_0^{*},{ \mathrm{a}}_0^{*},{ \mathrm{b}}_0^{*}$—value of sample colour components measured after storage.

The differences in perceivable colour (ΔE*) were classified as not noticeable (0–0.5), slightly noticeable (0.5–1.5), noticeable (1.5–3.0), very visible (3.0–6.0), and great (6.0–12.0) [24,25].

2.3.5. Texture

Cheese firmness was determined as force (in N) by using a TA-XTplus Texture Analyzer (Stable Micro Systems, Surrey, UK) with probe SMS P/1S. The maximum force (in N) was detected at the deformation rate (10 mm s⁻¹) and distance (10 mm) of the cheese.

2.4. Sensory Evaluation

Sensory analysis was conducted after cheese ripening (day 45) and at the end of storage (day 205). The evaluation took place in a sensory room with a trained panel of 7 members (both sexes, ages 20 to 50 years). The panellists were selected and trained according to the guidelines of ISO 8586:2012 [26]. Before assessment, samples were coded with 3-digit randomized numbers and served at room temperature. Cheese samples (5 × 2 × 2 cm blocks) were randomly presented to the panel members on identical plastic plates. The sensory evaluation scorecard, based on Bodyfelt et al. (1988) [27], allocated 50 points for flavour, 40 points for body and texture, and 10 points for appearance, with an overall maximum score of 100 points.

2.5. Microbiological Analysis

For microbiological analysis, viable counts of mesophilic LAB, Enterobacteriaceae spp., coliforms, yeasts, and moulds were determined in triplicate on the selective media for each species as described by Mileriene et al. (2021) [28]. A 10 g sample of cheese was mixed with 90 mL of sterile saline solution and homogenized with a Stomacher 400 Circulator (Seward, Worthing, UK). The suspensions were appropriately diluted and plated on a selective medium. LAB counts were enumerated on MRS agar (Oxoid, Basingstoke, UK) and M17 incubated at 30 °C for 72 h according to ISO 15214:1998 [29]. Yeasts and moulds were enumerated on Potato Dextrose agar (Oxoid, UK) at 25 °C for 120 h according to ISO 6611:2004 [30]. Coliforms were enumerated on Violet Red Bile agar (Liofilchem, Roseto degli Abruzzi, Italy) at 30 °C for 24 h according to ISO 4832:2006 [31]. Enterobacteria were enumerated on Violet Red Bile Glucose Agar (Liofilchem, Italy) at 37 °C for 24 h according to ISO 21528-2:2017 [32].

2.6. Statistical Analysis

All data processing and analysis was performed using SPSS statistical package (SPSS Inc., SPSS 24, Chicago, IL, USA). The data were subjected to one-way analysis of variance (ANOVA) with a confidence level of 95% (p ≤ 0.05). All the results in triplicate are expressed as the mean ± standard deviation.

3. Results and Discussion

3.1. Gouda Cheese Protection

3.1.1. Physicochemical Profile of Biocoated Gouda Cheese

Both single-day and treatment factors and their interaction had a significant effect on cheese colour, pH, water activity, moisture, and texture. However, cheese weight loss was influenced only by the time factor, while LAB CFU was affected by single-day and treatment factors. Fungi and enterobacteria CFU were influenced only by treatment (

Table 2. Effect of day, treatment, and their effect on cheese colour, acidity, moisture, a_w, texture, and weight loss, as well as sensory evaluation and microbiological parameters.

	L*	a*	b*	ΔE*	pH	aw	Moisture	Texture	Sensory Evaluation	Weight Loss	LAB	Mould	Yeast	Enterobacteria
Day	0.001	0.001	0.001	0.001	0.001	0.001	0.001	0.001	0.05	0.001	0.001	-	-	-
Treatment	0.001	0.014	0.001	0.001	0.001	0.001	0.001	0.001	-	0.243	0.001	0.001	0.05	0.001
Day × Treatment	0.001	0.018	0.001	0.003	0.001	0.001	0.001	0.001	-	0.988	0.29	-	-	-

). A summary of the physicochemical profile of Gouda cheese is presented in

Table 3. The effect of biopackaging and edible coating on physicochemical parameters of cheese during ripening and refrigerated storage.

Parameter	Sample	Day 1	Day 15	Day 30	Day 45	Day 205
Moisture	Bp6 + Ch + Coating	33.82 ± 0.01 Aa	37.68 ± 0.01 Ba	35.17 ± 0.01 Ca	34.73 ± 0.01 Da	39.05 ± 0.01 Eb
	Bp6 + Ch + Coating + Lp	33.89 ± 0.01 Aa	33.99 ± 0.01 Bb	35.2 ± 0.01 Cb	33.51 ± 0.01 Db	37.13 ± 0.08 Eacde
	Bp6 + Ch + Coating + Lh	30.7 ± 0.01 BCDEb	34.88 ± 0.01 AEc	34.23 ± 0.01 ABDEc	35.32 ± 0.01 ACEc	38.55 ± 0.51 ABCDbd
	Bp6 + Ch	35.49 ± 0.79 CEc	34.82 ± 0.01 CDEd	33.4 ± 0.01 ABDEd	36.39 ± 0.01 BCEd	39.5 ± 0.45 ABCDbc
	Pp + Ch	36.85 ± 0.01 Ad	30.89 ± 0.01 Be	33.03 ± 0.01 Ce	33.69 ± 0.01 De	39.46 ± 0.64 Eb
Weight loss	Bp6 + Ch + Coating	-	0.08 ± 0.05 Ea	0.12 ± 0.06 AEc	0.14 ± 0.06 AEa	1.39 ± 0.01 ABCDcde
	Bp6 + Ch + Coating + Lp	-	0.05 ± 0.01 Aa	0.07 ± 0.01 Bb	0.1 ± 0.01 Ca	1.45 ± 0.01 Dde
	Bp6 + Ch + Coating + Lh	-	0.06 ± 0.01 Ea	0.1 ± 0.01 Aa	0.13 ± 0.01 Aa	1.84 ± 0.01 ABCDde
	Bp6 + Ch	-	0.06 ± 0.01 Aa	0.09 ± 0.01 Bb	0.13 ± 0.01 Ca	1.14 ± 0.01 Dabcde
	Pp + Ch	-	0.04 ± 0.03 DEa	0.1 ± 0.07 Ea	0.15 ± 0.08 ABEa	1.64 ± 0.01 ABCDabcde
pH	Bp6 + Ch + Coating	5.43 ± 0.03 CDEa	5.49 ± 0.01 DEa	5.5 ± 0.01 ADEbc	5.62 ± 0.01 ABCcb	5.62 ± 0.01 ABCde
	Bp6 + Ch + Coating + Lp	5.54 ± 0.01 BDEb	5.51 ± 0.01 ACDEa	5.55 ± 0.01 BDEbc	5.59 ± 0.01 ABCc	5.59 ± 0.01 ABCde
	Bp6 + Ch + Coating + Lh	5.54 ± 0.01 BCDEb	5.49 ± 0.01 ACDEa	5.62 ± 0.01 ABa	5.64 ± 0.01 ABb	5.61 ± 0.01 ABde
	Bp6 + Ch	5.56 ± 0.03 EBb	5.54 ± 0.01 EBa	5.59 ± 0.03 AEBab	5.61 ± 0.01 AEBcb	5.68 ± 0.01 ABabc
	Pp + Ch	5.5 ± 0.01 BDEc	5.63 ± 0.01 ACEb	5.52 ± 0.01 BDEab	5.6 ± 0.01 ACEcb	5.68 ± 0.02 abc
aw	Bp6 + Ch + Coating	0.870 ± 0.001 BCDEa	0.880 ± 0.001 ACEc	0.884 ± 0.001 ABDEca	0.879 ± 0.001 ACEa	0.843 ± 0.007 ABCDcde
	Bp6 + Ch + Coating + Lp	0.865 ± 0.001 BCDEb	0.878 ± 0.001 ACEb	0.885 ± 0.001 ABDEca	0.876 ± 0.001 ACEb	0.843 ± 0.02 ABCDc
	Bp6 + Ch + Coating + Lh	0.858 ± 0.001 BCDEc	0.877 ± 0.001 ACDEb	0.888 ± 0.001 ABEedba	0.889 ± 0.001 ABEabe	0.834 ± 0.01 ABCDEabcde
	Bp6 + Ch	0.863 ± 0.02 BCDEd	0.881 ± 0.001 AEc	0.883 ± 0.001 ADEa	0.891 ± 0.001 ABCEabe	0.857 ± 0.008 ABCDac
	Pp + Ch	0.888 ± 0.001 BCDEe	0.878 ± 0.001 ADEb	0.880 ± 0.001 ADEedcb	0.883 ± 0.001 ABCEc	0.856 ± 0.01 ABCDac
L*	Bp6 + Ch + Coating	75.33 ± 1.74 BCDEc	70.14 ± 0.97 Aba	69.12 ± 1.39 Abd	68.88 ± 0.18 Ade	69.88 ± 0.01 Aa
	Bp6 + Ch + Coating + Lp	72.8 ± 2.01 CDEba	72.17 ± 0.31 CDEabe	68.39 ± 1.09 ABb	69.03 ± 1.01 ABd	68.11 ± 0.07 ABb
	Bp6 + Ch + Coating + Lh	69.33 ± 2.95 Aeba	71.33 ± 0.28 Aa	70.91 ± 1.2 Ad	69.66 ± 1.97 Ad	68.27 ± 0.16 Ac
	Bp6 + Ch	77.92 ± 3.23 Adc	73.28 ± 2.5 Be	71.59 ± 1.09 Bbd	73.38 ± 1.55 Babc	68.94 ± 0.05 Bd
	Pp + Ch	79.07 ± 1.51 BCDEdc	73.73 ± 1.34 ACe	69.95 ± 1.78 ABbd	72.4 ± 2.54 Aab	72.35 ± 0.01 Ae
a*	Bp6 + Ch + Coating	−2.538 ± 0.87 Aa	−1.85 ± 0.33 Abd	−1.47 ± 0.5 Aa	−2.12 ± 0.77 Aa	−2.43 ± 0.22 Aa
	Bp6 + Ch + Coating + Lp	−2.71 ± 1.26 Aa	−2.19 ± 0.43 Ab	−1.67 ± 0.14 Aa	−2.41 ± 0.45 Aa	−2.39 ± 0.64 Aa
	Bp6 + Ch + Coating + Lh	−2.47 ± 0.47 Aa	−1.36 ± 0.87 Abd	−1.23 ± 0.12 Aa	−2.16 ± 0.46 Aa	−1.23 ± 1.29 Aab
	Bp6 + Ch	−1.69 ± 0.37 DEa	−1.24 ± 0.08 Dd	−1.46 ± 0.37 Da	−3.31 ± 0.96 ABCEa	−0.5 ± 0.24 ADb
	Pp + Ch	−2.14 ± 0.42 Ea	−1.83 ± 0.63 Ebd	−2.45 ± 0.44 Eb	−2.94 ± 0.75 BEa	−0.24 ± 0.14 ABCDb
b*	Bp6 + Ch + Coating	25.92 ± 0.76 Ab	24.72 ± 3.07 Aabd	23.76 ± 0.7 Ad	21.18 ± 5.55 Aa	19.93 ± 0.07 Aa
	Bp6 + Ch + Coating + Lp	32.84 ± 2.36 BCDEa	24.11 ± 0.35 ACa	29.27 ± 1.11 ABDEe	21.81 ± 0.86 ACEa	25.05 ± 0.02 ACDb
	Bp6 + Ch + Coating + Lh	29.33 ± 2.27 BDEab	26.09 ± 1.03 ADabd	27.22 ± 1.32 De	21.09 ± 2.07 ABCEa	24.81 ± 0.02 ADc
	Bp6 + Ch	32.7 ± 4.47 Aa	23.78 ± 0.74 Ba	28.03 ± 1.74 ABe	23.04 ± 1.2 Ba	32.24 ± 0.02 Ad
	Pp + Ch	26.9 ± 1.63 Db	27.19 ± 1.4 Ddb	27.81 ± 1.81 De	23.26 ± 1.24 ABCEa	29.67 ± 0.01 De
Colour change (∆E*)	Bp6 + Ch + Coating	-	5.95 ± 2.54 Aa	6.89 ± 2.54 Aa	9.03 ± 4.53 Aa	7.77 ± 1.64 Acb
	Bp6 + Ch + Coating + Lp	-	9.19 ± 2.47 ABa	6.32 ± 2.55 Aa	11.96 ± 2.38 Ba	9.84 ± 1.72 ABc
	Bp6 + Ch + Coating + Lh	-	4.24 ± 3.22 Da	3.62 ± 2.86 Db	9.06 ± 2.16 ADa	4.48 ± 1.14 Db
	Bp6 + Ch	-	7.21 ± 2.09 Aa	5.53 ± 1.46 Aa	8.11 ± 3.06 Aa	6.85 ± 2.19 Acb
	Pp + Ch	-	6.01 ± 2.11 Aa	9.52 ± 2.63 Ac	7.81 ± 3.01 Aa	7.66 ± 1.53 Acb
Texture (N)	Bp6 + Ch + Coating	63.56 ± 7.33 CDabc	57.50 ± 10.15 CDb	116.78 ± 16.03 ABDEd	32.87 ± 4.07 ABCdb	44.38 ± 3.5 Cc
	Bp6 + Ch + Coating + Lp	67.3 ± 11.31 BDECabc	42.45 ± 13.33 Cbe	97.39 ± 14.71 BDEde	50.73 ± 18.14 Cd	56.56 ± 23.55 Ccde
	Bp6 + Ch + Coating + Lh	87.14 ± 18.45 BDEba	36.33 ± 10.09 ACbe	93.45 ± 9.26 BDEde	31.11 ± 12.69 ACdb	21.35 ± 2.73 ACab
	Bp6 + Ch	52.52 ± 15.44 Dc	33.45 ± 6.33 Ce	66.75 ± 16.76 BDEe	23.5 ± 5.89 ACb	31.20 ± 3.19 Cb
	Pp + Ch	52.75 ± 13.85 Cc	46.22 ± 11.38 Cbe	123.73 ± 31.47 ABDEd	40.94 ± 11.27 Cdb	33.57 ± 4.75 Cb

Different lower-case letters mean significant difference between samples in the same parameter (p ≤ 0.05). Different uppercase letters mean significant difference between days in the same parameter (p ≤ 0.05).

.

3.1.2. Moisture Content

Controlling moisture during cheese processing has a technical connotation for final cheese quality [33]. The moisture content in blank and coated Gouda cheese was evaluated throughout the ripening and storage periods (Figure 2). We detected lower moisture content in all coated samples at the beginning of the experiment (day 1), with results being on average 0.5% lower in plain coated cheese (Bp6 + Ch + Coating) and even lower in L. paracasei (Bp6 + Ch + Coating + Lp)- and L. helveticus (Bp6 + Ch + Coating + Lh)-supplemented cheese samples, 2.5 and 3%, respectively (p ≤ 0.001). Between both blank cheeses, those packed in Pp were moister compared to the Bp6-packed cheeses on day 1 (p = 0.008). During ripening, both blank samples’ moisture content dropped and rose again by the end of storage. This tendency was less expressed in coated samples. By the end of storage, blank samples retained the most moisture, plain coating contributed to moisture retention, and strain-incorporated coated cheese samples were least moist compared to the rest of the samples (p ≤ 0.001). This lower moisture content could be related to the drying-off period that was applied to all coated samples as well as the barrier properties of the packaging materials. Furthermore, the enhanced moisture absorption in the coated samples is likely to be attributed to pectin (good free water binder) incorporation into the LAWP-based film [34]. According to Dhall (2013) [35], edible coatings based on polymers, such as alginate and mucilages, are excellent barriers against gas exchange, but do not prevent the loss of moisture of the product to the medium, since they are hydrophilic coatings. In the present study, coatings without additives had superior water retaining properties compared to the strain-supplemented coatings, most potentially due to the high degradation of the coating matrix by the added LAB strains [21].

The influence of the strain on the moisture content of cheese was controversial: at the beginning, L. helveticus-supplemented samples were the least moist, while at the end of ripening and storage, they were moister compared to L. paracasei-supplemented ones (p < 0.05). The activity of living strains in the coating on the cheese matrix contributing to moisture gain or loss should be considered. Hassanien et al. (2014) [36] state that lactic acid, produced by LAB, helps to draw out moisture from the cheese mass, thus intensifying the drying-off of the product. According to Ye et al. (2018) [37], living strains in the coating have a negative effect on its barrier properties due to the destruction of intermolecular interactions in the solution during film formation and increases in the volume of voids, thereby slightly decreasing the tensile strength of the films, but significantly increasing water vapour permeability. In contrast to our results, Lactococcus lactis L3A21M1 and Lc. garvieae SJM17 incorporated into an edible fresh-cheese coating composed of alginate, maltodextrin, and glycerol reduced moisture and weight losses of fresh cheeses during storage [17]. These changes in the film may not be so visible in coating applications on fresh cheese.

3.1.3. Weight Loss

As Gouda cheese ages, it gradually loses moisture through evaporation, contributing to weight loss. No statistical differences among samples in weight loss were observed during the ripening period. However, we determined a significant weight loss in all cheese samples throughout the storage period (Figure 3; (p ≤ 0.01)). Surprisingly, all coated samples lost the most weight by the end of storage, and even more with L. helveticus added, compared to the weight loss in blank samples packed in biodegradable packaging (Bp6 + Ch; p ≤ 0.05). We suspect that L. helveticus enhanced protein hydrolysis, and due to this consequence, more water-soluble proteins were formed in the process. The conventionally packed samples lost more weight than those packed in Bp6 packaging, except L. helveticus-supplemented samples (p ≤ 0.05). In addition to this, there is evidence that lactic acid bacteria supplementation in cheese can increase microstructure rupture within cheese matrix, due to excreted enzymes and their activity, resulting in a looser network structure [38], thus allowing unbound water molecules to evaporate more easily.

3.1.4. pH

The overall pH deviation was assessed at every time point of ripening and at the end of storage to determine the flux direction of acidity, which is known to influence microbial growth (Figure 4). In the beginning of the study, on day 1, cheese with plain coating demonstrated the lowest pH value compared to the rest of the samples, while the strain-incorporated coated cheese and blank cheese in Bp6 packaging expressed the highest pH values (p ≤ 0.01). On day 15, all the coated cheese samples had a lower pH compared to all blank samples on day 15. A slow constant increase in pH was determined in the coated and blank biopackaged samples during the ripening period. At the end of the ripening period, there was no difference in cheese pH, except that the strain-incorporated coated cheese samples differed in pH—L. paracasei in the coating expressed lower pH than L. helveticus (p = 0.04). In comparison to the coated samples, the blank samples in both forms of packaging altered towards a neutral pH that was observed at the end of storage (p ≤ 0.05). Regarding the conventionally packed Pp cheese samples, a wave-like pH change was seen with a sharp pH increase from 5.55 to 5.68, down to 5.61 by day 30 and up again to 5.75 on day 45. According to Diezhandino et al. (2016) [39], such dynamic in pH values could result from low-molecular-weight compounds being released during proteolysis and lipolysis occurring in cheese during ripening, corresponding to more expressed cheese flavour, characteristic of Gouda cheese. Based on the present study results, the influence of the so-called buffering effect of LAWPC coating could be considered. Whey protein is amphoteric, meaning it can act as both an acid and a base. Depending on the pH of the cheese, the whey protein in the coating may buffer changes in acidity, helping to stabilize the pH to some extent. Additionally, the whey protein-based coating could act as a barrier, limiting the diffusion of ions, gases, and water vapour between the cheese and its environment, slowing down the acidification process caused by lactic acid bacteria during ripening. As a result, the pH changes may be slightly delayed in cheese with this type of coating compared to uncoated cheese. This observation correlates to the studies of other authors, confirming that coating application has a pH-altering effect in a different direction and only to a small extent [40,41].

3.1.5. Water Activity

During the ripening process of hard cheeses, water activity undergoes changes that impact the texture, flavour, and overall quality of the cheese. The water activity of Gouda cheese itself has been reported as an average of 0.972 [42]. We detected higher water activity (Figure 5) in both blank cheese samples on day 1 and on day 205 compared to the coated samples (p ≤ 0.05). Conventionally packed samples expressed higher a_w than those packed in Bp6 in the beginning and at the end of experiment (p ≤ 0.005). From day 15, a_w increased in all biopacked samples and decreased in conventionally packed ones (p ≤ 0.05) throughout the ripening time, with Bp6 + Ch and Bp6 + Ch + Coating + Lh demonstrating the highest water activity, while samples packed in Pp (Pp + Ch) had less free water, and the rest of the coated samples—Bp6 + Ch + Coating and Bp6 + Ch + Coating + Lp—had even less (p ≤ 0.005). The correlation analysis between a_w and moisture content revealed significant variations across different samples. All coated samples demonstrated stronger correlations (Bp6 + Ch + Coating: r = 0.51, p = 0.08; Bp6 + Ch + Coating + Lp: r = 0.65, p = 0.02; Bp6 + Ch + Coating + Lh: r = 0.90, p < 0.001) compared to their uncoated counterparts. Among the coated samples, the strongest and most significant correlations were observed in strain-containing samples, with the strongest correlation found in the Bp6 + Ch + Coating + Lh sample (r = 0.90, p < 0.001). In contrast, all uncoated samples (Bp6 + Ch, Pp + Ch) exhibited weak or no correlations (r = 0.13, p = 0.55; r = 0.10, p = 0.64, respectively). This indicates that the coating participates in reducing moisture content and a_w in cheese during ripening. The addition of microbial strains in the coating plays a crucial role, leading to more intense evaporation of moisture and consequently lowering a_w. The findings of other researchers are controversial and highly depend on the type of coating, cheese, and environment. In contrast to our findings, Silva et al. (2022) [17] state that the presence of a coating could slow down the ripening process by limiting the exchange of gases and moisture between the cheese and its environment, helping to maintain a more consistent moisture, texture, and flavour while reducing the risk of spoilage due to excessive microbial growth. According to Jafarzadeh et al. (2021) [43], coatings and films may act as a physical barrier against mould growth on the cheese’s surface that can contribute to water activity changes, and by preventing mould development, the water activity remains more stable. All the coatings in the present study helped to slow down the mould growth in biopacked cheese (Section 3.3. Microbiological Profile of Biocoated Gouda Cheese), demonstrating lower a_w by the end of ripening. Between the two strains used for incorporation into the coating, L. helveticus demonstrated the highest variation in water activity. In the beginning and at the end of storage, it was at its lowest, increasing significantly during ripening compared to rest of the samples. Conformably, due to the lowest a_w in L. helveticus-supplemented coated samples, yeast counts were below detectable levels in these samples by the end of storage (Section 3.3. Microbiological Profile of Biocoated Gouda Cheese).

3.1.6. Colour Change

The application of an edible coating on cheese can influence its colour coordinates and colour change during storage. These changes depend on the type of coating material, its transparency, and its interactions with the cheese matrix. In the present study, edible coatings, applied on Gouda cheese, affected the colour of cheese and contributed to general colour change in cheese (Table 2, p = 0.001). Edible coatings, especially strain-supplemented ones, decreased the L* value and made the cheese appear darker throughout the ripening period (Figure 6), compared to the brighter blank cheese samples (p ≤ 0.001). Both packaging forms were able to protect blank cheese from darkening throughout the ripening period, although it became darker by the end of storage, especially in Bp6 packaging (p ≤ 0.001). We detected no difference in L* value between strain-supplemented coated samples throughout the ripening period. However, by the end of storage supplementation with L. helveticus, lighter tones were added to the coated cheese samples compared to the plain coating (p ≤ 0.05).

The a* value, representing the position on the red–green axis (Figure 7), was significantly influenced by the application of coatings on cheese (Table 2, p = 0.0018). On day 1, all coated cheese samples expressed more green hues than their blank counterparts (p ≤ 0.01). By the end of ripening, all of them shifted the cheese’s colour towards more red hues, compared to both blank samples (p ≤ 0.001). However, at the end of storage, blank samples, especially those conventionally packed, were redder in colour compared to the coated ones (p ≤ 0.001). This phenomenon might be associated with the greenish colour of the LAWPC-based coating and the ability of added LAB strains to synthesize B vitamins that impact the green colour of the product [44].

The results of b* values (Figure 8) were more controversial compared to L* and a* values on day 1. We observed a variation in the samples around the yellow–blue axis throughout the ripening period. Only the cheese coated with plain coating expressed a steady change from yellow to blue hues throughout the experiment. Strain-supplemented coated samples and blank samples packed in Bp6 were more yellow than the rest of the samples (p ≤ 0.05). By the end of ripening and storage, all coated samples shifted towards negative values indicating blueness, while blank samples shifted the cheese’s colour towards more yellow hues (p ≤ 0.001).

The impact of the strain on coated cheese colour was significant (p ≤ 0.05). L. helveticus-supplemented coated samples were detected as more light, red, and blue by the end of ripening and storage, compared to darker L. paracasei-supplemented coatings shifting cheese colour tones to more green and yellow. Additionally, L. paracasei-supplemented coated cheese samples demonstrated higher colour change (∆E*, calculated by comparing the L*a*b* values on day 1 with the values on days 15, 30, 45, and 205, Figure 9) compared to L. helveticus-supplemented samples (p ≤ 0.001) while no significant colour change was observed among other samples during ripening and storage. This may indicate the unique strain and cheese matrix interaction such as acid production, proteolysis, lipolysis, pigment interaction, the production of metabolites, or other colour-contributing components, leading to changes in the colour of the cheese. Moreover, Ye et al. (2018) [37] report that the addition of LAB can change the spatial structure of molecules, destroy intermolecular interactions, and increase the intermolecular space, which can reduce the barrier properties of the coating, contributing to colour change.

In relation to our previous study on liquid whey protein (LWPC)-coated acid curd cheese [28], our latest findings lead to the conclusion that the combination of coating and packaging has no or a less expressed effect on colour changes or differences than that has been stated by authors that applied the coating on package-free cheese [21,45]. In addition, measuring coating effects on the colours of fresh-cut cheese samples could be beneficial since the cheese would not be affected by chemical changes and would not alter in structure and appearance during ripening and storage, and initial effects could be observed [46].

3.1.7. Texture

Gouda cheese is known for its creamy and smooth texture. During ripening, enzymes present in the cheese break down the protein and fat, which results in a softer and smoother texture, which were observed in the present study (Figure 10) by the end of ripening in all cheese samples (p ≤ 0.05). On the other hand, moisture reduction led to the formation of a firmer texture in the cheese [47]. Conformably, blank Gouda cheese samples in the beginning of the experiment and by the end of storage expressed softer textures compared to the coated ones (p ≤ 0.001). Among the coated samples, L. helveticus-supplemented ones were firmer in the beginning of the experiment, while by the end of ripening and storage, they became more tender. On the contrary, L. paracasei-supplemented samples demonstrated a relatively softer texture at the beginning that became firmer by the end of ripening (p ≤ 0.05). Applied plain coating significantly contributed to the changes in cheese texture, making it firmer at the beginning and at the end of storage (Bp6 + Ch + Coating). In contrary to our findings, reductions in water loss (~10%, by weight), hardness, and discolouration were observed after the application of whey protein-based coating to hard cheese [45]. The LAWPC–pectin-based coating applied to the cheese packed in biodegradable packaging led to firmer texture and slightly more moisture loss when combined with biodegradable packaging.

3.2. Sensory Evaluation

Texture and flavour are closely linked in cheese. As Gouda ripens, it develops a more complex and concentrated flavour profile. The interplay of flavours contributes to the overall experience of the cheese, complementing its evolving texture [48]. Edible coatings can enhance the visual appeal of cheeses by providing a smooth and uniform surface. Coated cheeses may have a more attractive and glossy appearance compared to uncoated cheeses, which can be particularly beneficial for marketing and consumer appeal. The coating can influence the texture of the cheese by providing a protective layer on the surface. Depending on the coating’s characteristics, it can create a thin, flexible film that affects the mouthfeel and texture perception. Some coatings may contribute to a smoother, creamier, or softer texture, while others may add a slight crunch or firmness [49]. Edible coatings are generally designed to be tasteless and odourless to avoid interfering with the cheese’s natural flavour. However, some coatings might have a slight influence on flavour perception due to interactions with volatile compounds in the cheese. The impact on flavour is typically minimal, and coatings are formulated to maintain the cheese’s original taste [17,28]. In the present study, no impact of treatment or treatment-by-day interaction was detected during ripening and storage on any sensory parameters (p > 0.05,

Table 4. Sensory parameters of Gouda cheese during ripening and storage.

Parameter	Sample	Day 45	Day 205
Flavour	Bp6 + Ch + Coating	7.2 ± 1.84 aA	7.7 ± 1.41 aA
	Bp6 + Ch + Coating + Lp	6.8 ± 1.84 aA	8.03 ± 1.01 aA
	Bp6 + Ch + Coating + Lh	5.8 ± 1.98 aA	8.23 ± 1.11 aA
	Bp6 + Ch	5.2 ± 2.4 aA	8.13 ± 0.99 aA
	Pp + Ch	4.35 ± 2.62 aA	7.15 ± 0.07 aA
Body and texture	Bp6 + Ch + Coating	7.4 ± 1.27 aA	6.97 ± 1.41 aA
	Bp6 + Ch + Coating + Lp	6.65 ± 2.2 aA	8.33 ± 0.95 aA
	Bp6 + Ch + Coating + Lh	7 ± 1.13 aA	8.5 ± 0.78 aA
	Bp6 + Ch	6.75 ± 0.78 aA	7.87 ± 1.91 aA
	Pp + Ch	5.35 ± 1.9 aA	8 ± 1.13 aA
Appearance	Bp6 + Ch + Coating	7.85 ± 1.49 aA	8.33 ± 1.04 aA
	Bp6 + Ch + Coating + Lp	7.9 ± 1.41 aA	8.37 ± 1 aA
	Bp6 + Ch + Coating + Lh	7.8 ± 1.56 aA	8.53 ± 0.84 aA
	Bp6 + Ch	7.9 ± 1.41 aA	8.3 ± 1.15 aA
	Pp + Ch	7.8 ± 1.23 aA	8.35 ± 1.63 aA
Total acceptability	Bp6 + Ch + Coating	7.35 ± 1.56 aA	7.47 ± 0.96 aA
	Bp6 + Ch + Coating + Lp	6.85 ± 1.94 aA	8.19 ± 0.96 aA
	Bp6 + Ch + Coating + Lh	6.48 ± 1.6 aA	8.37 ± 0.84 aA
	Bp6 + Ch	6.09 ± 1.66 aA	8.04 ± 1.36 aA
	Pp + Ch	5.1 ± 2.2 aA	7.61 ± 0.58 aA

Different lower-case letters mean significant difference between samples in the same parameter (p ≤ 0.05). Different uppercase letters mean significant difference between days in the same parameter (p ≤ 0.05).

). The single-day factor (Table 2) significantly affected cheese flavour, body and texture, and total acceptability (p ≤ 0.01), indicating an increase in sensory perception during storage. However, according to our panellists’ comments, the cheeses samples packed in biodegradable packaging expressed milder flavour, characteristic of young cheese, while conventionally packed samples demonstrated flavour and texture characteristic of ripe Gouda-type cheese. It seems that the chosen packaging delays the ripening of cheese. The addition of the strains to the coating slightly, yet noticeably, enhanced the flavour of the cheese, adding strain-specific notes to the traditional Gouda-type cheese flavour profile. This is in agreement with our previous studies [21,22]. To our knowledge, there are no other available studies analyzing the impact of living strains incorporated into the coating on the sensory characteristics of cheese.

3.3. Microbiological Profile of Biocoated Gouda Cheese

During the ripening and storage of Gouda cheese, the LAB CFU changed significantly, contributing to lactose breakdown and enzyme synthesis, which are essential for flavour development, texture changes, and preservation [36]. In the present study, individual-day and treatment factors significantly affected the LAB CFU in cheese (p ≤ 0.001), with no impact of their interaction (p = 0.29) (Table 2). We observed low CFU of LAB during the first 15 days, while from 15 to 45 days, the LAB CFU increased and remained at the same level until the end of ripening (p ≤ 0.01) (Table 5). In comparison to the uncoated samples, slightly higher CFU of LAB was detected in all coated samples at the beginning of ripening (p > 0.05). In agreement with our results, Ramos et al. (2012) [45] demonstrate that the storage of cheeses coated with an WP film blended with a mix of antimicrobial substances inhibited the growth of contaminant or pathogenic microorganisms, while permitting lactic acid bacteria to grow normally during storage.

As expected, cheese with strain-incorporated coating demonstrated significantly higher CFU of LAB throughout ripening and storage (p ≤ 0.05), while less LAB were found in the samples with plain coating (p ≤ 0.05). Between the two strain-incorporated coatings, the LAB count increased rapidly with L. helveticus throughout storage (p ≤ 0.01). The survival of the strain in the coating during ripening and prolonged storage of cheese is important in ensuring the protective effect on the cheese. Regarding our previous study [21] and the evidence of Gerez et al. (2012) [50], the incorporation of probiotic strains in a pectin–whey protein matrix could be an effective solution, protecting the strain from unfavourable acidic conditions in LAWPC-based coating. It has been stated that pectin fibre has a protective effect on intact cells, while LAWPC contains small amounts of reducing sugars, providing adenosine triphosphate (ATP), and improving the bacteria’s survival [51]. We observed a significant decline in LAB CFU at the end of storage (p ≤ 0.001), due to a reduction in available nutrients in the cheese’s matrix and due to lower refrigeration temperature, which resulted in a reduction in metabolic activity. Interestingly, we detected higher counts of LAB in samples packed in Bp6 compared to the samples packed in conventional Pp packaging (p ≤ 0.05).

Gouda cheese, like any other food product, is susceptible to spoilage by various microorganisms. The development of spoilage microorganisms in Gouda cheese can occur due to factors such as improper cheese packaging and storage conditions and contamination during processing, with a few concerning cheese contaminators such as Enterobacteriaceae spp. and fungi [1,52].

In the present study, we detected fungi and enterobacteria by the end of the ripening and storage period. Their amounts were significantly influenced by the applied treatment (Table 2, p ≤ 0.05). Normally, the cheese-making process and technological aids, along with proper hygienic conditions, are designed to control the growth of undesirable or harmful moulds and yeast. The introduction of new biodegradable packaging in the present study negatively interfered with established control mechanisms, contributing to increased mould at the end of ripening (Table 5). No fungi were detected in the present study during 30 days of ripening in any sample. On day 45, we detected mould in every sample, with double counts prevalent in blank biopacked samples (Bp6 + Ch) compared to the rest of the samples (p ≤ 0.05). The plain and lactobacilli-incorporated coatings were able to keep the mould at bay until the end of ripening; their counts did not differ significantly from control cheese packed in conventional Pp plastic. There were no significant differences in mould CFU among different tested LAWPC coatings; however, the mould CFU dropped in cheese samples with the L. helveticus-incorporated coating (Bp6 + Ch + Coating + Lh; p ≥ 0.05). Accordingly, the addition of the L. helveticus strain to the coating reduced the mould count by approximately 2 log₁₀ CFU/g, compared to the one-fold reduction in cheese with plain and L. paracasei-incorporated coatings. Some lactic acid bacteria strains can produce antimicrobial agents, which can develop strong inhibitory activity against many microorganisms, including those with spoilage and pathogenic effects, due to different mechanisms, providing a preservation effect on perishable cheese products [53].

In the cheese-making process, there is a possibility for cheese to be contaminated with enterobacteria. However, during the ripening process, most cheeses, including Gouda, undergo a combination of factors such as pH decrease, influence of added salt, and competition from beneficial bacteria and their metabolites; furthermore, the addition of preservatives in cheese milk or the treatment of the cheese surface with preservatives eliminates the growth of fungi, which can inhibit the growth of undesirable flora. In our study, Enterobacteriaceae spp. was found only in blank samples on day 45, with higher counts detected in conventionally packed cheese (Pp + Ch; p ≤ 0.04). Coated samples did not show any presence. We speculate that the organic acids and other postbiotics present in the plain LAWPC coating had a destructive effect on some spoilage bacteria, but only to a certain extent (during ripening).

During refrigerated storage of Gouda cheese, the growth of fungi is typically slowed down due to the lower temperatures. However, some fungi can still survive in the cheese, especially if the storage conditions are not optimal or if the cheese is not adequately protected from environmental contaminants; for example, if the cheese is not adequately wrapped or sealed, there is a possibility that fungi may continue to grow, especially on the surface of the cheese. Only yeasts were detected at the end of storage at 4 °C in the present study, with their counts not exceeding 2 log₁₀ CFU/g. On average, both conventional (Pp + Ch) and biopackaging (Bp6 + Ch) materials demonstrated similar counts of yeast in all samples; however, the variation in the mean was higher in conventionally packed cheese. Interestingly, plain coating significantly contributed to the growth of yeast (p ≤ 0.05), while both strain-supplemented coatings were able to control their growth. However, yeast CFU were lower in the L. helveticus strain-incorporated coated (Bp6 + Ch + Coating + Lh) cheese samples (p ≤ 0.05). The yeast CFU being below detectable levels in L. helveticus-incorporated coated samples shows that yeasts are susceptible to L. helveticus metabolites. The strain-dependant antimicrobial effects of various coating applications were confirmed by other authors. According to Hua et al. (2022) [54], probiotic-fortified coating significantly inhibited the proliferation of psychrophilic bacteria (Pseudomonas spp.), Enterobacteriaceae, and spiked Listeria monocytogenes on salmon fillets during refrigeration storage. Accordingly, the application of coating with incorporated Lactococcus sp. cells on cheeses significantly reduced (p < 0.05) contamination by Listeria monocytogenes on the surface and prevented the growth of mesophilic bacteria by the sixth and eighth day of storage at 4 °C [17]. The study of Cheong et al. (2014) [55] confirms our theory, where the antifungal effect of LAB was shown on several mould species, P. commune being one of them. WP films combined with probiotics can also be utilized for their antimicrobial activity. The incorporation of Lactobacillus sakei NRRL B-1917 in WP films decreased the count of L. monocytogenes by 1.4 log₁₀ CFU g⁻¹ after 120 h, while E. coli were reduced by 2.3 log₁₀ CFU g⁻¹ during 36 h of refrigerated storage [56].

Table 5. The effect of biopackaging and edible coating on lactic acid bacteria, fungi, and Enterobacteriaceae sp. counts in cheese during ripening and refrigerated storage.

Parameter	Sample	Day 1	Day 15	Day 30	Day 45	Day 205
LAB, log10 CFU g−1	Bp6 + Ch + Coating	8.58 ± 0.05 CDbcd	9.33 ± 0.62 Ec	9.69 ± 0.06 AEc	9.69 ± 0.04 AEbce	8.27 ± 0.37 BCDde
	Bp6 + Ch + Coating + Lp	8.65 ± 0.09 BCDEb	9.75 ± 0.18 AEce	9.81 ± 0.0.4 AEc	9.84 ± 0.09 AEac	8.29 ± 0.07 ABCDde
	Bp6 + Ch + Coating + Lh	8.80 ± 0.06 BCDEb	10.19 ± 0.03 AEe	10.16 ± 0.0.6 AEd	10.14 ± 0.0.3 AEabde	8.25 ± 0.18 ABCDabde
	Bp6 + Ch	7.76 ± 0.71 BCDdc	9.68 ± 0.05 AEce	9.76 ± 0.13 AEc	9.83 ± 0.04 AEc	8.39 ± 0.27 BCDab
	Pp + Ch	8.32 ± 0.28 BCDbcd	9.64 ± 0.19 AEce	9.80 ± 0.01 AEc	9.85 ± 0.03 AEac	8.07 ± 0.01 BCDab
Yeast, log10 CFU g−1	Bp6 + Ch + Coating					2.12 ± 0.05 c
	Bp6 + Ch + Coating + Lp					1.40 ± 0.94 b
	Bp6 + Ch + Coating + Lh					0.39 ± 0.12 a
	Bp6 + Ch					1.36 ± 0.10 b
	Pp + Ch					1.31 ± 0.87 b
Mould, log10 CFU g−1	Bp6 + Ch + Coating				2.50 ± 0.03 a
	Bp6 + Ch + Coating + Lp				2.39 ± 0.35 a
	Bp6 + Ch + Coating + Lh				1.91 ± 0.12 b
	Bp6 + Ch				3.40 ± 0.25 c
	Pp + Ch				1.69 ± 0.39 b
Enterobacteriaceae, log10 CFU g−1	Bp6 + Ch + Coating
	Bp6 + Ch + Coating + Lp
	Bp6 + Ch + Coating + Lh
	Bp6 + Ch				0.58 ± 0.64 a
	Pp + Ch				1.32 ± 0.28 b

Different lower-case letters mean significant difference between samples in the same parameter (p ≤ 0.05). Different uppercase letters mean significant difference between days in the same parameter (p ≤ 0.05).

Table 5. The effect of biopackaging and edible coating on lactic acid bacteria, fungi, and Enterobacteriaceae sp. counts in cheese during ripening and refrigerated storage.

Parameter	Sample	Day 1	Day 15	Day 30	Day 45	Day 205
LAB, log10 CFU g−1	Bp6 + Ch + Coating	8.58 ± 0.05 CDbcd	9.33 ± 0.62 Ec	9.69 ± 0.06 AEc	9.69 ± 0.04 AEbce	8.27 ± 0.37 BCDde
	Bp6 + Ch + Coating + Lp	8.65 ± 0.09 BCDEb	9.75 ± 0.18 AEce	9.81 ± 0.0.4 AEc	9.84 ± 0.09 AEac	8.29 ± 0.07 ABCDde
	Bp6 + Ch + Coating + Lh	8.80 ± 0.06 BCDEb	10.19 ± 0.03 AEe	10.16 ± 0.0.6 AEd	10.14 ± 0.0.3 AEabde	8.25 ± 0.18 ABCDabde
	Bp6 + Ch	7.76 ± 0.71 BCDdc	9.68 ± 0.05 AEce	9.76 ± 0.13 AEc	9.83 ± 0.04 AEc	8.39 ± 0.27 BCDab
	Pp + Ch	8.32 ± 0.28 BCDbcd	9.64 ± 0.19 AEce	9.80 ± 0.01 AEc	9.85 ± 0.03 AEac	8.07 ± 0.01 BCDab
Yeast, log10 CFU g−1	Bp6 + Ch + Coating					2.12 ± 0.05 c
	Bp6 + Ch + Coating + Lp					1.40 ± 0.94 b
	Bp6 + Ch + Coating + Lh					0.39 ± 0.12 a
	Bp6 + Ch					1.36 ± 0.10 b
	Pp + Ch					1.31 ± 0.87 b
Mould, log10 CFU g−1	Bp6 + Ch + Coating				2.50 ± 0.03 a
	Bp6 + Ch + Coating + Lp				2.39 ± 0.35 a
	Bp6 + Ch + Coating + Lh				1.91 ± 0.12 b
	Bp6 + Ch				3.40 ± 0.25 c
	Pp + Ch				1.69 ± 0.39 b
Enterobacteriaceae, log10 CFU g−1	Bp6 + Ch + Coating
	Bp6 + Ch + Coating + Lp
	Bp6 + Ch + Coating + Lh
	Bp6 + Ch				0.58 ± 0.64 a
	Pp + Ch				1.32 ± 0.28 b

Different lower-case letters mean significant difference between samples in the same parameter (p ≤ 0.05). Different uppercase letters mean significant difference between days in the same parameter (p ≤ 0.05).

4. Conclusions

This experiment revealed that vacuum-sealed biopackaging demonstrated lesser barrier properties and provided less protection against microbiological spoilage than conventional plastic. Based on the results, it can be concluded that plain coating applied on cheese for additional protection controls Enterobacteriaceae sp. growth and reduces fungal (up to 1.0 log₁₀ CFU/g) contamination in Gouda cheese packed in biodegradable packaging. The primary decrease was detected on day 45 of ripening and towards Enterobacteriaceae most efficiently. The spoilage-preventing qualities of the plain coating stands out in terms of less complexity and may be exploited for microbiological quality control during cheese ripening in biodegradable packaging.

The addition of L. helveticus to the coating solution protected cheese from fungus growth during prolonged cold storage. Consequently, packing a high-lipid food product—Gouda cheese—into protective strain-enriched edible coating could be a good strategy as it has nearly no influence on the physical–chemical parameters of the cheese and ensures food safety with active compounds, certainly even longer than the shelf-life period. For further studies, supernatants of protective strains or their bacteriocins can be used to simplify the coating formulation. Additionally, the components chosen for in vivo protection of packed cheese are entirely safe for use in the food.

During the ripening process, Gouda undergoes several changes that contribute to its characteristic texture and flavour. It is important to note that the ripening process and sensory characteristics in our study were negatively influenced by biopackaging material. According to our observation, the length of ripening has to be longer for the tested biopackaging materials to achieve the distinct textures and flavour experiences of traditional Gouda cheese.