Effect of Lactic Acid Bacteria on Nutritional and Sensory Quality of Goat Organic Acid-Rennet Cheeses

Abstract

The aim of this study was to evaluate the applicability of selected Lactobacillus strains, previously isolated from spontaneously fermented foods, as starter cultures in the production of organic dairy products—acid-rennet goat’s cheeses under industrial conditions. The basic composition and the effect of starter cultures on the physicochemical, microbiological, sensory as well textural properties during the production and storage of goat’s cheese were evaluated. Lactic acid bacteria count in cheese samples was at a high level of about 8 log CFU/g. The cheeses made with Levilactobacillus brevis B1 and Lactiplantibacillus plantarum Os2 bacterial cultures additions have showed more favorable Lipid Quality Indices than for the control one with the addition of acid whey. The time of ripening of the cheeses significantly (p < 0.005) changed their consistency—they became softer and more elastic and less moist. It is possible that the selected cultures of L. brevis B1 and L. plantarum Os2 isolated from traditional cheeses can be successfully applied to goat’s milk cheese production. The strain L. brevis B1 is highly recommended as a starter culture for goat’s milk cheese production, taking into account the good microbiological and sensory quality as well as the chemical composition.

1. Introduction

In the last 60–80 years, agriculture has moved from traditional, natural plant cultivation and animal husbandry, used for thousands of years, to very intensive, often non-organic production [1,2,3,4]. Taking into account the latest findings on the effect of microbiota abundance and biodiversity on human health, it is claimed that the solution that may help avoid the plague of civilization diseases, which can be now observed, would be a harmonized diet based on organically produced, minimally processed, not sterilized food, coming from the areas where our ancestors lived for generations. Scientific research confirms that the microbiome is flexible enough to change as the environment changes. This phenomenon contributes to the ability of organisms to respond and adapt to environmental changes that can occur quicker than traditional body adaptation processes [5].

Developing food products with selected starter cultures is one way to improve the health status of consumers, as well as the texture and taste of the food, thereby improving its properties and reducing production costs. Moreover, the use of an appropriate starter culture can influence the nutritional composition, safety and overall quality of food. Some authors emphasize the health-promoting properties of specific environmental lactic acid bacteria and their effect on human health [6].

One of the promising raw materials of growing consumer interest, providing nutritional benefits, is goat’s milk and its dairy products. Goat’s milk, compared to cow’s milk, contains higher amounts of calcium, magnesium and phosphorus [7,8] and has fat globules of a smaller size which facilitate digestion [9]. In addition, the oligosaccharides present in goat’s milk are not only more concentrated, but also have a profile similar to human milk [10,11]. Goat’s milk is often recommended for the prevention of cardiovascular diseases and allergies and is also used to stimulate the immune system. Goat products have a low allergenic potential, therefore, they are a good alternative for consumers who are sensitive or allergic to certain proteins present in cow’s milk [12]. Goat’s milk can be regarded as a natural functional food. The regular consumption of goat’s milk and its products should be encouraged [9,13].

Goat’s milk cheese is a highly valuable product, being a good source of protein, macronutrients, organic acids, vitamins and many other components of our diet. According to Santurino et al. [14], the consumption of goat’s cheese naturally enriched with n-3 PUFA and CLA could play a potential role as a high nutritional health-enhancing food. It is also a potential source of bioactive peptides and a rich set of microorganisms, especially lactic acid bacteria, which have a positive effect on the microbiome and human health [15].

The role of starter cultures in the cheese production process is fairly well understood. The addition of bacteria as a starter plays a crucial role in cheese production technology. It aims to direct the fermentation processes during the processing of the cheese mass and inhibits the development of undesirable microbiota. During the ripening of the cheese mass, due to numerous factors, complicated biochemical processes take place, such as protein proteolysis, fat lipolysis, lactose glycolysis and the transformation of citrate. Protein hydrolysis is caused by the presence of enzymes (proteases) from the milk itself, coagulating preparations and bacteria. As a result of the processes, as the cheese matures, smaller protein fractions, such as peptides and free amino acids are formed. Fat lipolysis plays a role in developing the taste and aroma of the end product. During maturation, lipolytic enzymes hydrolyze the fat into short-chain and volatile fatty acids. The type of starting microbiota and the maturation time determine the level of lipid changes. As a result of lactic acid bacteria fermentation, the lactose contained in the milk is converted into lactic acid. The accumulation of lactic acid leads to a decreased pH value, protects against the development of undesirable microorganisms and helps with syneresis. The transformation of lactose and citrate contributes to the formation of compounds such as diacetyl, acetoin, ethanol and acetates [16,17,18].

Taking into account the above findings, the aim of this study was to evaluate the applicability of selected Lactobacillus strains, previously isolated from spontaneously fermented foods, as starter cultures in the production of organic dairy products—acid-rennet goat’s cheeses—under industrial conditions and to examine the microbiological, nutritional and sensory quality of the obtained cheeses during two-month maturation storage. The idea arose from an emerging need for the restoration of the population’s intestinal microbiota. Using the unique, selected environmental strains of lactic acid bacteria with potential probiotic properties for the production of organic goat’s cheese can enrich the microbiota of consumers with valuable strains of environmental bacteria in the form of high-nutritional-value food products.

2. Materials and Methods

2.1. Bacteria Strains and Starter Cultures Preparation

Bacterial strains have been obtained from the internal collection of microorganisms of the Chair of Food Hygiene and Quality Management SGGW-WULS in Warsaw: Levilactobacillus brevis B1—isolated from “bundz”—and Lactiplantibacillus plantarum Os2—isolated from “oscypek”, traditional cheeses made in Tatra mountain. The previous studies demonstrated their good technological as well as potentially probiotic properties [19,20,21]. These bacterial strains were selected at the preliminary stage of research, according to enzymatic activity (lipases, proteases and peptidases), the ability to ferment lactose into lactic acid, as well as sensory properties of fermented goat’s milk.

The starter cultures were prepared as follows: bacteria were grown on de Man, Rogosa and Sharpe (MRS, LabM, Bury, UK) broth at 37 °C for 24 h to a concentration of 10⁹ CFU/mL, then centrifuged (6000× g, 10 min) and washed in sterile PBS (phosphate-buffered saline 1×, pH 7.4) in triplicate. Next, bacterial cells were resuspended in 1 L of goat’s milk and again incubated at 37 °C for 24 h, obtaining an inoculum concentration of 10⁸ CFU/mL.

2.2. Cheese Samples

The research material consisted of acid-rennet cheeses made from goat’s milk (Carpathian breed). The acid-rennet cheeses were produced in a small family’s organic farm from the Podkarpackie Province in Poland. As a control sample, acid-rennet goat’s cheese with the addition of acid whey (symbol AW) was used. The acid whey was obtained from organic cottage cheese production in the same organic farm and was used as a “natural” starter of bacteria cultures. The research samples were goat’s acid-rennet cheeses with the addition of L. brevis B1 strain (symbol B1) and L. plantarum Os2 (symbol Os2).

To raw, unpasteurized goat’s milk, acid whey (8% w/v) or starter culture of B1 or Os2 strains (2% w/v) were added. Next, the goat’s milk was left at room temperature, left for a few hours, then heated in pots to 40 °C and microbial rennet was added. After 20–40 min, a clot (called a clag) formed on the surface, which was stirred with a ferula or by hand. After about 5 min, a small amount of boiling water was poured onto the surface, causing the curd to settle. In the meantime, appropriate forms were prepared, made of stainless steel and lined with cheese cloths. The appropriate amount of the curd was placed on the scarves, ironed and then the lids were put on and secured. In addition, the entire closed mold was poured over with boiling water in order to “close” the cheese. Pressing the cheese took about 4–6 h and then the cheese was brined in a saturated salt solution for 6–8 h. Two series of goat’s cheese were made under industrial conditions.

The cheese samples (approx. 1 kg) were vacuum packed and stored at chilled temperature (4 °C) for 2 months of maturation. Once a month, the samples were taken and chemical, microbiological, textural and sensory analyses were made to determine the quality changes during storage.

2.3. Microbiological Analysis

The spread plate technique was used to determine the count of microorganisms. To determine the count of microorganisms of lactic acid bacteria (LAB) the following microbiological media and methodology tests were used: MRS agar (LabM, Heywood, UK) in combination with incubation under anaerobic conditions (AnaeroGen System, Oxoid, Hampshire, UK) was applied in accordance with the ISO15214:2002 [22], to determine the number of rods from the Enterobacteriaceae family (ENT), MacConkey agar No. 3 (LabM, Heywood, UK) in accordance with the ISO 21528-2 [23] and to determine the number of yeasts and molds (YM), YGC agar (Sabouraud Dextrose with chloramphenicol lab Agar, Biomaxima, Lublin, Poland) in accordance with the ISO 21527-1 [24] and ISO 21527-2 [25]. The number of microorganisms was expressed as the logarithm of the colony forming units per gram (log CFU/g).

The presence of selected pathogenic bacteria was determined using the enrichment culture method with the media indicated in the standards—XLD agar (Xylose Lysine Deoxycholate Agar, LabM, UK) and RAPID’ Salmonella agar (Bio-Rad, Hecules, CA, USA)—to determine the presence of Salmonella, in accordance with the ISO 6579-1 [26], and ALOA agar (Listeria according to Ottaviani and Agosti Agar, Bio-Rad, Hecules, CA, USA) and PALCAM agar (LabM, Heywood, UK) to determine the presence of Listeria monocytogenes, in accordance with the ISO 11290-2 [27].

2.4. Chemical Composition

The selected parameters of water, protein, fat, lactose, NaCl and phosphorus content of the goat’s cheeses were determined after production. The tests were performed in 4 repetitions.

• The water content [%] was determined by the weight method according to PN-ISO 1442:2000 [28]. The measured samples were dried at 103 °C for 30 min.
• The protein content was done using the Kjeldahl method according to PN-A-04018:1975/Az3:2002 [29]. The method consists of determining the total nitrogen content, subsequently using the conversion factor of nitrogen content into protein content (for meat = 6.25).
• The content of free fat was made by the Weibull–Berntrop gravimetric method according to PN ISO 8262-3:2011 [30].
• Composition of fatty acids was determined by the gas chromatography (GC) method with flame ionization detection (HP 6890 II) according to PN-EN ISO 5508:1996 [31]. A BPX 70 high-polar capillary column (60 m × 0.25 mm, 25 μm) was used for ester separation. Analysis conditions: column temperature programmable in the range of 140–210 °C, injector temperature: 210 °C, detector temperature: 250 °C, carrier gas: helium. The tests were performed in 4 repetitions immediately after the production process, after 1 and 2 months of storage. The values are given in %.
• The Lipid Quality Indices were examined after the production process, after 1 and 2 months of storage. The indices were calculated using the following formulas [32,33,34,35,36]:
  The Index of Atherogenicity

  AI = (C12:0 + (4 × C14:0) + C16:0)/(Σ n-3 PUFA + Σ n-6 PUFA + Σ MUFA),

  (1)
  Index of Thrombogenicity

  TI = (C14:0 + C16:0 + C18:0)/[(0.5 × C18:1) + (0.5 × other MUFA) + (0.5 × Σ n − 6 PUFA) + (3 × Σ n − 3 PUFA) + Σ n − 3 PUFA/Σ n − 6 PUFA)],

  (2)
  Hypocholesterolemic Fatty Acids

  DFA = UFA + C18:0,

  (3)
  Hypercholesterolemic Fatty Acids

  OFA = C12:0 + C14:0 + C16:0,

  (4)
  The ratio of hypocholesterolemic and hypercholesterolemic fatty acids

  H/H = (C18:1 n-9 + C18:2 n-6 + C18:3 n-3)/(C12:0 + C14:0 + C16:0),

  (5)
  Health-promoting index

  HPI = Σ UFA/[C12:0 + (4 × C14:0) + C16:0],

  (6)
• Cholesterol content was made by the extraction of the lipid fraction from the cheese sample, esterification of fatty acids and derivatization of cholesterol in the presence of an internal standard. The sample was analyzed by GC method with flame ionization detection. The tests were performed in 4 repetitions immediately after the production process, after 1 and 2 months of storage. The cholesterol value is expressed in mg/100 g of product.
• Lactose content was performed by liquid chromatography (LC) using an RID refractive index detector. Lactose was separated and compared to the external standard method. The lactose value was expressed in mg/100 g.
• The phosphorus content was determined as total phosphorus content [%], expressed as P₂O₅, included the mineralization of the sample, precipitation of phosphorus in the form of choline phosphoromolybdate and weight determination of total phosphorus, according to PN-A-82060:1999 [37].
• The chloride content was determined using the potentiometric method according to PN-ISO 1841-2:2002 [38].

2.4.1. Water Activity

The water activity (aw) of cheese samples was determined by AQUALAB Pawkit Water Activity Meter (METER Group, Inc., Washington, DC, USA) according to the ISO 18787:2017 [39]. The samples were placed in special, closed vials, immediately after opening the package of cheese samples, and maintained at temperature of 25 °C for 1 h. The result is given as the value of the water activity with the standard deviation of three replications.

2.4.2. Determination of the Oxidation-Reduction Potential (ORP)

A 30 g sample of the previously ground product was transferred to a porcelain mortar. The cheese was ground thoroughly with a pestle, with addition of several small portions of distilled water (30 cm³) at temperature of 40 °C in until a homogeneous emulsion was obtained. The resulting emulsion was adjusted to a temperature of 20 °C and ORP value was measured with a SevenCompactTM S220 (Mettler Toledo, Columbus, OH, USA) with an InLab Redox Pro electrode and expressed in mV. The tests were performed in 4 repetitions immediately after the production process, after 1 and 2 months of storage.

2.4.3. Acidity-pH Value

The measurement was made with the SevenCompactTM S220 device (Mettler Toledo, OH, USA) with an InLab electrode. The tests were performed in 4 repetitions immediately after the production process, after 1 and 2 months of storage. The results were presented with 0.01 accuracy with standard deviations.

2.4.4. Titratable Acidity

A 5 g sample of cheese of the previously ground product was transferred to a porcelain mortar. The cheese was thoroughly ground with a pestle, gradually adding 50 cm³ of distilled water at temperature of 40 °C in small portions until obtaining a homogeneous emulsion. After that, 2 cm³ of 2% alcoholic phenolphthalein solution was added and titrated with 0.25 M NaOH until a slightly pink color persisted for 30 s. The tests were performed in 4 repetitions immediately after the production process, after 1 and 2 months of storage. The values are given in °SH.

2.5. Sensory Evaluation

The sensory quality of the acid-rennet goat’s cheese was determined using the scaling method [40]. The linear scale (100 mm) was converted to numerical values (0–10 c. u.). A set of cheese samples were presented to the assessors. Before proper evaluation, the distinguishing attributes were chosen, and in preliminary session their understanding was established and defined. The following set of attributes were established for the evaluation: 5 attributes of odor (milk’s fermentation, goat’s milk, fatty, sharp, mature cheese), 1 attribute of color (color intensity) and 3 texture attributes (softness, moisture, elasticity). Due to the severity of SARS-CoV-2 disease, it was necessary to eliminate the assessment of flavor attributes. It was assumed that flavor sensations in food are highly influenced by the aroma and texture compounds. It is well known from literature that the volatile compounds and their composition that occur in food products can affect the specific aroma of foods and the flavor of the resulting products [41]. The anchor marks of the tested attributes ranged from none (the left side) to very high intensity (the right side of the scale).

The cheese samples were portioned (average piece size of about 15 g) and put into disposable containers manufactured from food-safe plastic. The containers were then closed and individually coded with 3-digit codes and given in random order. The samples were assessed after production, after 1 month and after 2 months of storage.

The trained panel consisted of 8 members (7 women and 1 man, age 30–58), who were extensively and formally tested before being selected, according to the ISO standard (ISO 8586:2012) [42]. The assessors had 4 to 18 years of practical experience with sensory procedures and sensory evaluation of different food products with different methods. The panelists’ ability to differentiate product samples was verified by various concentrations of volatile and non-volatile stimuli.

The evaluators had the conditions for full concentration provided. The ambient temperature was 22–23 °C. The assessment and condition mode were established in accordance with Meilgaard et al. [43].

2.6. Instrumental Determination of Color

To determine the color, the CR-300 Chroma-Meter colorimeter (Konica Minolta, Tokyo, Japan) was used. The determinations were made under diffused illumination at 0° and a diaphragm diameter of 8 mm. For the determination, cut slices of cheese with a thickness of 20 mm were used. The apparatus was calibrated before use and calibration of the spectrophotometer was performed on a white standard. The color was measured in the CIE L*a*b* system. The tests were performed in 20 repetitions immediately after the production process, after 1 and 2 months of storage.

2.7. Instrumental Texture Evaluation

Texture profile analysis (TPA) was accomplished with CT3 Texture Analyzer (Brookfield Ametek, Middleborough, MA, USA) instruments. The speed of compression of cheese samples were 0.50 mm/s and the compression was made twice up to 50% of their original height with a cylindrical head with a diameter of 38.1 mm and a height of 20 mm with a 10,000 g maximum pressing force. The tested cheese had the shape of a cube with a side of 20 mm. The following textural parameters were determined: hardness 1 (force necessary to attain a given deformation; N), hardness 2 (force necessary to attain a given deformation; N), adhesiveness (work necessary to overcome the attractive forces between the surface of the food and the surface of the other materials with which the food comes into contact; mJ), cohesiveness (extent to which a material can be deformed before it ruptures; adimensional), springiness (rate at which a deformed material goes back to its undeformed condition after the deforming force is removed: mm), gumminess (energy required to disintegrate a semi-solid food to a state ready for swallowing: a product of a low degree of hardness and a high degree of cohesiveness; N) and chewiness (energy required to masticate a solid food to a state ready for swallowing: a product of hardness, cohesiveness and springiness; mJ) [44]. The measurements were carried out at room temperature, 20 min after the samples were taken out of the cooling chamber. The test was carried out in 6 replications.

2.8. Mathematical and Statistical Analysis of the Results

Two independent experimental series (batches) of the product were manufactured under industrial conditions. The measurements were repeated several times for each batch of the product. A one-way analysis of variance (ANOVA) at p < 0.05 was used, first to test the treatments effect (AW, B1, Os2) and then between the storage time (0, 1, 2 months). Means and standard deviations were calculated. The Tukey’s test was conducted for a mean comparison (p < 0.05). The normality of distribution of all analyzed traits was checked by Shapiro–Wilk test. The Statistica 13.1 program (StatSoft Inc., Tulsa, OK, USA) was used to perform the calculations.

3. Results

3.1. Microbiological Quality

The microbiological quality of the cheese samples is presented in

Table 1. Microbiological quality of the cheese samples during storage [log CFU/g].

Count/Presence of Microorganisms [log CFU/g]	Time [Month]			Cheese Symbol
	0	1	2
LAB	8.11 Aa ± 0.01	8.04 Aa ± 0.07	7.83 Aa ± 0.07	AW
ENT	5.90 Aa ± 0.01	4.39 Ba ± 0.16	4.37 Ba ± 0.01
YM	3.68 Aa ± 0.01	4.69 Ba ± 0.21	4.65 Ba ± 0.07
SALM	nd	nd	Nd
LIST	nd	nd	Nd
LAB	7.86 Ab ± 0.01	7.93 Ba ± 0.01	7.82 ABa ± 0.13	B1
ENT	5.57 Aa ± 0.09	5.16 ABa ± 0.18	4.36 Ba ± 0.01
YM	3.45 Aa ± 0.01	4.29 ABa ± 0.30	4.94 Ba ± 0.02
SALM	nd	nd	Nd
LIST	nd	nd	Nd
LAB	8.08 Aa ± 0.01	8.09 Aa ± 0.07	7.82 Aa ± 0.16	Os2
ENT	6.66 Ab ± 0.03	4.89 Ba ± 0.17	4.70 Bb ± 0.01
YM	3.63 Aa ± 0.01	4.65 Ba ± 0.04	4.81 Ca ± 0.05
SALM	nd	nd	Nd
LIST	nd	nd	Nd

Explanatory: AW—farmer’s acid-rennet goat’s cheese with acid whey, B1—farmer’s acid-rennet goat’s cheese with L. brevis B1, Os2—farmer’s acid-rennet goat’s cheese with L. plantarum Os2; LAB—the number of lactic acid bacteria; ENT—the number of Enterobacteriaceae; YM—the number of yeasts and molds; SALM—Salmonella sp.; LIST—Listeria monocytogenes; nd—not detected in 25 g of sample. The values were expressed as means ± SD; means in the same row followed by different uppercase letters within the same sample at different times are significantly different (p < 0.05); means in the same column followed by different lowercase letters within samples at the same time are significantly different (p < 0.05); (n = 3).

.

The lactic acid bacteria (LAB) count was at a high level of about 8 log CFU/g and was similar in all samples, however, the LAB count changed only in the B1 sample during storage (p < 0.05). The count of Enterobacteriaceae bacteria was in the range of 5–6 log CFU/g immediately after production and decreased during storage (p < 0.05) to approximately 4–5 log CFU/g in all samples. The initial yeast and mold contamination of the goat’s cheeses was low, approx. 3 log CFU/g, and increased after storage (p < 0.05) in all of the cheese samples. No pathogenic bacteria (L. monocytogenes and Salmonella) were found in any of the tested cheese samples.

3.2. Chemical Composition

The basic composition, NaCl content and phosphorus content were different in the produced goat’s cheeses (

Table 2. The basic composition of acid-rennet goat’s cheeses directly after production.

	Cheese Symbol
Composition	AW	B1	Os2
Water [%]	46.60 A ± 0.00	48.10 B ± 0.14	47.35 C ± 0.07
Protein [%]	26.50 C ± 0.28	25.60 B ± 0.00	23.85 A ± 0.07
Fat [%]	20.45 A ± 0.49	19.55 A ± 0.07	21.9 B ± 0.14
NaCl [%]	1.36 B ± 0.01	1.32 A ± 0.00	1.31 A ± 0.01
Phosphorus [%]	1.34 B ± 0.01	1.36 B ± 0.04	1.21 A ± 0.01
Lactose [mg/100 g]	nd	nd	nd

Explanatory: AW—goat’s cheese with acid whey, B1—goat’s cheese with L. brevis B1, Os2—goat’s cheese with L. plantarum Os2; nd—not detected. The values were expressed as means ±SD; means in the same row followed by different uppercase letters represent significant differences (p < 0.05) (n = 4).

). The water content was in the range 46.60% to 48.10%. The protein content ranged from 23.85% for the Os2 cheese to 26.50% for the AW control cheese. In terms of water and protein content, all cheeses differed significantly (p < 0.05). The fat content in the AW and B1 cheeses was similar (p > 0.05) and amounted to 19.55–20.45%. The Os2 cheese (21.9%) had a significantly higher fat content. The NaCl content was similarly ranged from 1.31 to 1.36%, with the AW control cheese having a slightly higher content. The Os2 cheese had the lowest (p < 0.05) phosphorus content (1.21%). In all the tested samples, no lactose was found after cheese samples production (Table 2), which proves the high ability of the used LAB strains to ferment this sugar.

The most common fatty acids in goat’s cheeses were presented in

Table 3. Selected fatty acid composition, sums and cholesterol content in goat’s acid-rennet cheeses after production, and after 1 and 2 months of storage.

Parameter	Time [Month]			Cheese Symbol
	0	1	2
Σ SFA [%]	71.75 Cb ± 0.07	71.43 Ca ± 0.10	70.98 Cc ± 0.44	AW
Σ MUFA [%]	23.80 Aa ± 0.00	24.05 Ab ± 0.06	24.30 Ac ± 0.20
Σ PUFA [%]	4.45 Ab ± 0.07	3.43 Aa ± 0.05	3.50 Aa ± 0.00
Trans [%]	2.90 Aa ± 0.00	3.00 Bb ± 0.00	2.98 Ab ± 0.05
Omega-3 [%]	1.20 Aa ± 0.00	1.20 Aa ± 0.00	1.20 Aa ± 0.00
Omega-6 [%]	2.25 Aa ± 0.07	2.13 Ab ± 0.05	2.20 Ab ± 0.00
Omega-9 [%]	17.30 Aa ± 0.00	17.25 Aa ± 0.06	17.15 Aa ± 0.10
Cholesterol [mg/100 g of product]	51.10 Ba ± 1.56	49.08 Ba ± 1.09	59.85 Ab ± 0.00
Σ SFA [%]	68.20 bA ± 0.00	66.73 aA ± 0.17	67.05 aA ± 0.42	B1
Σ MUFA [%]	26.90 aC ± 0.00	27.20 bC ± 0.08	27.50 bC ± 0.45
Σ PUFA [%]	4.90 aC ± 0.00	5.03 bC ± 0.10	5.10 bC ± 0.14
Trans [%]	3.10 aB ± 0.00	3.20 aC ± 0.00	3.18 aB ± 0.13
Omega-3 [%]	1.30 aB ± 0.00	1.40 bC ± 0.00	1.45 bB ± 0.10
Omega-6 [%]	2.60 Cb ± 0.00	2.45 Ca ± 0.06	2.53 Cb ± 0.05
Omega-9 [%]	20.10 Ca ± 0.00	20.05 Ca ± 0.06	20.18 Ca ± 0.33
Cholesterol [mg/100 g of product]	48.30 Aa ± 1.27	47.55 Aa ± 3.63	62.98 Ab ± 2.60
Σ SFA [%]	69.00 Bc ± 0.14	68.70 Bb ± 0.08	68.45 Ba ± 0.13	Os2
Σ MUFA [%]	26.20 Ba ± 0.14	26.20 Ba ± 0.08	26.30 Ba ± 0.14
Σ PUFA [%]	4.80 Ba ± 0.00	4.80 Ba ± 0.00	4.88 Bb ± 0.05
Trans [%]	2.85 Aa ± 0.07	2.93 Aa ± 0.05	2.93 Aa ± 0.05
Omega-3 [%]	1.30 Ba ± 0.00	1.30 Ba ± 0.00	1.43 Bb ± 0.05
Omega-6 [%]	2.50 Bb ± 0.00	2.30 Ba ± 0.00	2.35 Ba ± 0.06
Omega-9 [%]	19.65 Bc ± 0.07	19.38 Bb ± 0.05	19.18 Ba ± 0.10
Cholesterol [mg/100 g of product]	54.45 Ca ± 1.77	53.75 Ba ± 0.37	74.88 Bb ± 2.63

Explanatory: AW—goat’s cheese with acid whey, B1—goat’s cheese with L. brevis B1, Os2—goat’s cheese with L. plantarum Os2; Σ SFA—all saturated fatty acids; Σ MUFA—all monounsaturated fatty acids; Σ PUFA—all polyunsaturated fatty acids; trans—all trans fatty acids. The values were expressed as means ± SD; means in the same row followed by different uppercase letters within the same sample at different times are significantly different (p < 0.05); means in the same column followed by different lowercase letters within samples at the same time are significantly different (p < 0.05) (n = 4).

, while the full profile of fatty acids composition is available in the

Table A1. Fatty acid composition and cholesterol content in goat’s acid-rennet cheeses after production and after 1 and 2 months of storage (n = 4).

Parameter	Time [Month]
	0	1	2	0	1	2	0	1	2
C4:0 [%]	2.20 ± 0.00	2.13 ± 0.10	2.08 ± 0.10	2.10 ± 0.14	2.05 ± 0.06	2.13 ± 0.33	2.20 ± 0.00	2.08 ± 0.05	2.05 ± 0.06
C6:0 [%]	2.35 ± 0.07	2.28 ± 0.05	2.23 ± 0.05	2.20 ± 0.00	2.08 ± 0.05	2.05 ± 0.26	2.20 ± 0.00	2.13 ± 0.05	2.10 ± 0.00
C8:0 [%]	2.60 ± 0.00	2.53 ± 0.05	2.50 ± 0.00	2.25 ± 0.07	2.20 ± 0.00	2.08 ± 0.17	2.30 ± 0.00	2.30 ± 0.00	2.30 ± 0.00
C10:0 [%]	8.85 ± 0.07	8.88 ± 0.05	8.75 ± 0.06	7.40 ± 0.00	7.38 ± 0.05	6.95 ± 0.33	7.80 ± 0.00	7.83 ± 0.05	7.70 ± 0.08
C10:1 [%]	0.10 ± 0.00	0.10 ± 0.00	0.20 ± 0.00	0.10 ± 0.00	0.10 ± 0.00	0.10 ± 0.00	0.10 ± 0.00	0.10 ± 0.00	0.10 ± 0.00
C12:0 [%]	2.95 ± 0.07	3.00 ± 0.00	3.10 ± 0.00	2.50 ± 0.00	2.65 ± 0.06	2.55 ± 0.06	2.70 ± 0.00	2.80 ± 0.00	2.85 ± 0.06
C13:0 [%]	0.10 ± 0.00	0.10 ± 0.00	0.10 ± 0.00	0.10 ± 0.00	0.10 ± 0.00	0.10 ± 0.00	0.10 ± 0.00	0.10 ± 0.00	0.10 ± 0.00
C14:0 [%]	9.10 ± 0.00	9.03 ± 0.05	9.03 ± 0.05	8.75 ± 0.07	8.70 ± 0.00	8.50 ± 0.00	9.00 ± 0.00	9.00 ± 0.00	8.93 ± 0.05
C14:1 [%]	0.30 ± 0.00	0.30 ± 0.00	0.30 ± 0.00	0.30 ± 0.00	0.30 ± 0.00	0.30 ± 0.00	0.30 ± 0.00	0.30 ± 0.00	0.30 ± 0.00
C15:0 br [%]	0.40 ± 0.00	0.40 ± 0.00	0.40 ± 0.00	0.40 ± 0.00	0.40 ± 0.00	0.40 ± 0.00	0.40 ± 0.00	0.40 ± 0.00	0.40 ± 0.00
C15:0 [%]	0.80 ± 0.00	0.90 ± 0.00	0.90 ± 0.00	0.80 ± 0.00	0.85 ± 0.06	0.83 ± 0.05	0.80 ± 0.00	0.90 ± 0.00	0.90 ± 0.00
C15:1 [%]	0.20 ± 0.00	0.20 ± 0.00	0.20 ± 0.00	0.20 ± 0.00	0.20 ± 0.00	0.20 ± 0.00	0.20 ± 0.00	0.20 ± 0.00	0.20 ± 0.00
C16:0 [%]	28.85 ± 0.07	28.73 ± 0.15	28.35 ± 0.13	25.85 ± 0.07	25.50 ± 0.08	25.53 ± 0.22	26.65 ± 0.07	26.40 ± 0.00	26.20 ± 0.00
C16:1 [%]	0.80 ± 0.00	0.80 ± 0.00	1.08 ± 0.05	0.80 ± 0.00	0.80 ± 0.00	1.00 ± 0.08	0.85 ± 0.07	0.90 ± 0.00	1.10 ± 0.00
C17:0 br	0.80 ± 0.00	0.80 ± 0.00	0.80 ± 0.00	0.80 ± 0.00	0.83 ± 0.05	0.85 ± 0.06	0.80 ± 0.00	0.80 ± 0.00	0.90 ± 0.00
C17:0 [%]	0.60 ± 0.00	0.60 ± 0.00	0.60 ± 0.00	0.60 ± 0.00	0.60 ± 0.00	0.63 ± 0.05	0.60 ± 0.00	0.60 ± 0.00	0.60 ± 0.00
C17:1 [%]	0.20 ± 00	0.20 ± 0.00	0.20 ± 0.00	0.20 ± 0.00	0.20 ± 0.00	0.20 ± 0.00	0.20 ± 0.00	0.20 ± 0.00	0.20 ± 0.00
C18:0 [%]	11.85 ± 0.07	11.78 ± 0.05	11.85 ± 0.06	14.05 ± 0.07	13.93 ± 0.05	14.10 ± 0.41	13.15 ± 0.07	13.08 ± 0.05	13.03 ± 0.05
C18:1 trans [%]	2.90 ± 0.00	3.00 ± 0.00	2.98 ± 0.05	3.10 ± 0.00	3.20 ± 0.00	3.18 ± 0.13	2.85 ± 0.07	2.93 ± 0.05	2.93 ± 0.05
C18:1 cis9 [%]	17.20 ± 0.00	17.25 ± 0.06	17.15 ± 0.10	20.00 ± 0.00	20.05 ± 0.06	20.18 ± 0.33	19.55 ± 0.07	19.38 ± 0.05	19.18 ± 0.10
C18:1 cis11 [%]	0.70 ± 0.00	0.60 ± 0.00	0.60 ± 0.00	0.70 ± 0.00	0.70 ± 0.00	0.70 ± 0.00	0.70 ± 0.00	0.60 ± 0.00	0.60 ± 0.00
C18:1 c other [%]	1.30 ± 0.00	1.50 ± 0.00	1.50 ± 0.00	1.40 ± 0.00	1.55 ± 0.06	1.55 ± 0.06	1.35 ± 0.07	1.50 ± 0.00	1.60 ± 0.00
C18:2 [%]	1.95 ± 0.07	1.93 ± 0.05	2.00 ± 0.00	2.30 ± 0.00	2.25 ± 0.06	2.33 ± 0.05	2.20 ± 0.00	2.10 ± 0.00	2.25 ± 0.06
C18:3 n3 [%]	1.00 ± 0.00	1.00 ± 0.00	1.00 ± 0.00	1.10 ± 0.00	1.20 ± 0.00	1.18 ± 0.05	1.10 ± 0.00	1.10 ± 0.00	1.13 ± 0.05
C18:2 c9 t11 [%]	1.00 ± 0.00	1.00 ± 0.00	1.00 ± 0.00	1.00 ± 0.00	1.08 ± 0.05	1.03 ± 0.10	1.00 ± 0.00	1.10 ± 0.00	1.00 ± 0.00
C20:0 [%]	0.20 ± 0.00	0.20 ± 0.00	0.20 ± 0.00	0.20 ± 0.00	0.20 ± 0.00	0.28 ± 0.05	0.20 ± 0.00	0.20 ± 0.00	0.30 ± 0.00
C20:1 [%]	0.10 ± 0.00	0.10 ± 0.00	0.10 ± 0.00	0.10 ± 0.00	0.10 ± 0.00	0.10 ± 0.00	0.10 ± 0.00	0.10 ± 0.00	0.10 ± 0.00
C20:2 [%]	0.10 ± 0.00	0.10 ± 0.00	0.10 ± 0.00	0.10 ± 0.00	0.10 ± 0.00	0.10 ± 0.00	0.10 ± 0.00	0.10 ± 0.00	0.10 ± 0.00
C20:3 n6 [%]	0.10 ± 0.00	0.10 ± 0.00	0.10 ± 0.00	0.10 ± 0.00	0.10 ± 0.00	0.10 ± 0.00	0.10 ± 0.00	0.10 ± 0.00	0.00 ± 0.00
C20:4 n6 [%]	0.10 ± 0.00	0.10 ± 0.00	0.10 ± 0.00	0.10 ± 0.00	0.10 ± 0.00	0.10 ± 0.00	0.10 ± 0.00	0.10 ± 0.00	0.10 ± 0.00
C20:5 EPA [%]	0.10 ± 0.00	0.10 ± 0.00	0.10 ± 0.00	0.10 ± 0.00	0.10 ± 0.00	0.10 ± 0.00	0.10 ± 0.00	0.10 ± 0.00	0.10 ± 0.00
C22:0 [%]	0.10 ± 0.00	0.10 ± 0.00	0.10 ± 0.00	0.10 ± 0.00	0.10 ± 0.00	0.10 ± 0.00	0.10 ± 0.00	0.10 ± 0.00	0.10 ± 0.00
C22:5 n3 [%]	0.10 ± 0.00	0.10 ± 0.00	0.10 ± 0.00	0.10 ± 0.00	0.10 ± 0.00	0.18 ± 0.05	0.10 ± 0.00	0.10 ± 0.00	0.20 ± 0.00
SFA [%]	71.75 ± 0.07	71.43 ± 0.10	70.98 ± 0.44	68.20 ± 0.00	66.73 ± 0.17	67.05 ± 0.42	69.00 ± 0.14	68.70 ± 0.08	68.45 ± 0.13
MUFA [%]	23.80 ± 0.00	24.05 ± 0.06	24.30 ± 0.20	26.90 ± 0.00	27.20 ± 0.08	27.50 ± 0.45	26.20 ± 0.14	26.20 ± 0.08	26.30 ± 0.14
PUFA [%]	4.45 ± 0.07	3.43 ± 0.05	3.50 ± 0.00	4.90 ± 0.00	5.03 ± 0.10	5.10 ± 0.14	4.80 ± 0.00	4.80 ± 0.00	4.88 ± 0.05
Trans [%]	2.90 ± 0.00	3.00 ± 0.00	2.98 ± 0.05	3.10 ± 0.00	3.20 ± 0.00	3.18 ± 0.13	2.85 ± 0.07	2.93 ± 0.05	2.93 ± 0.05
Omega-3 [%]	1.20 ± 0.00	1.20 ± 0.00	1.20 ± 0.00	1.30 ± 0.00	1.40 ± 0.00	1.45 ± 0.10	1.30 ± 0.00	1.30 ± 0.00	1.43 ± 0.05
Omega-6 [%]	2.25 ± 0.07	2.13 ± 0.05	2.20 ± 0.00	2.60 ± 0.00	2.45 ± 0.06	2.53 ± 0.05	2.50 ± 0.00	2.30 ± 0.00	2.35 ± 0.06
Omega-9 [%]	17.30 ± 0.00	17.25 ± 0.06	17.15 ± 0.10	20.10 ± 0.00	20.05 ± 0.06	20.18 ± 0.33	19.65 ± 0.07	19.38 ± 0.05	19.18 ± 0.10
Cholesterol [mg/100 g of products]	51.10 ± 1.56	49.08 ± 1.09	59.85 ± 0.00	48.30 ± 1.27	47.55 ± 3.63	62.98 ± 2.60	54.45 ± 1.77	53.75 ± 0.37	74.88 ± 2.63
	AW			B1			Os2
	Cheese symbol

, Appendix A.

The main fatty acids present in the tested acid-rennet cheeses were palmitic C16:0, oleic C18:1cis9, stearic C18:0 and myristic C14:0. Fatty acids capric C10:0, caprylic C8:0, caproic C6:0 and butyric C4:0 were observed in smaller amounts. The sum of the saturated fatty acids (SFA) of the fresh cheeses was 68.20–71.75%, the sum of the monounsaturated fatty acids (MUFA) was approx. 23–26% and of the polyunsaturated (PUFA) was approx. 4.4–4.9%. A statistically significant (p < 0.05) decrease in the SFA content was observed after 1 and 2 months of storage, with a simultaneous significant increase in the content of MUFA and PUFA in the AW and B1 cheeses. In the case of the B1 and Os2 cheeses, a significant increase in omega-3 after storage was noted (p < 0.05). In the fresh samples, the cholesterol content was 51.10, 48.30 and 54.45 mg/100 g of the cheese (AW, B1 and Os2, respectively). A significant (p < 0.05) increase in the cholesterol content was observed in all trials after 1 and 2 months of storage, with the highest increase observed in the case of the Os2 cheese.

Moreover,

Table A2. The Lipid Quality Indices of goat’s cheeses after production and after 1 and 2 months of storage.

LQI	Time [Month]			Cheese Symbol
	0	1	2
AI	2.50 Bc ± 0.01	2.48 Bc ± 0.00	2.44 Ac ± 0.15	AW
TI	1.93 Ac ± 0.00	1.91 Ab ± 0.01	1.90 Ab ± 0.02
DFA	40.10 Aa ± 0.00	39.26 Aa ± 0.11	39.65 Aa ± 0.06
OFA	40.90 Cc ± 0.15	40.76 Bc ± 0.25	40.48 Ac ± 0.12
H/H	0.49 Aa ± 0.00	0.50 Aa ± 0.01	0.50 Aa ± 0.06
HPI	0.41 Aa ± 0.00	0.41 Aa ± 0.10	0.41 Aa ± 0.08
AI	2.06 Ba ± 0.02	2.03 Ba ± 0.00	1.97 Aa ± 0.17	B1
TI	1.67 Aa ± 0.01	1.62 Aa ± 0.12	1.60 Aa ± 0.05
DFA	45.85 Ac ± 0.15	46.16 Bc ± 0.02	46.70 Cc ± 0.05
OFA	37.10 Ca ± 0.00	36.85 Ba ± 0.05	36.58 Aa ± 0.12
H/H	0.63 Ab ± 0.02	0.64 Ac ± 0.01	0.65 Ab ± 0.05
HPI	0.50 Ab ± 0.01	0.51 Ab ± 0.06	0.53 Ac ± 0.00
AI	2.18 Ab ± 0.02	2.19 Ab ± 0.01	2.15 Ab ± 0.00	Os2
TI	1.71 Ab ± 0.01	2.59 Cc ± 0.01	2.51 Bc ± 0.09
DFA	44.15 Bb ± 0.05	44.08 Ab ± 0.06	44.21 Bb ± 0.23
OFA	38.35 Bb ± 0.05	38.20 Ab ± 0.00	37.98 Cb ± 0.12
H/H	0.60 Ab ± 0.01	0.60 Ab ± 0.01	0.60 Ab ± 0.05
HPI	0.47 Ab ± 0.13	0.48 Ab ± 0.02	0.48 Ab ± 0.00

Explanatory: AW—goat’s cheese with acid whey, B1—goat’s cheese with L. brevis B1, Os2—goat’s cheese with L. plantarum Os2; LQI—Lipid Quality Indices; AI—Index of atherogenicity, TI—Index of thrombogenicity, DFA—Hypocholesterolemic fatty acids, OFA—Hypercholesterolemic fatty acids, H/H—The ratio of hypocholesterolemic and hypercholesterolemic fatty acids; HPI—Health-promoting index. The values were expressed as means ± SD; means in the same row followed by different uppercase letters within the same sample at different times are significantly different (p < 0.05); means in the same column followed by different lowercase letters within samples at the same time are significantly different (p < 0.05) (n = 4).

in Appendix B shows the Lipid Quality Indices (LQI) for all types of cheeses immediately after production and after 1 and 2 months of refrigeration storage. Among the examined goat’s cheeses, the highest AI index was found in AW (2.50) and it was lower in Os2 (2.18) and B1 (2.06). The examined cheeses showed a low TI index, ranging from 1.67 (B1) to 1.93 (AW). In the case of the B1 and Os2 goat’s cheeses, a favorable share of DFA compared to OFA was observed. The AW goat’s cheese was characterized by a higher OFA index. The AW goat’s cheese was characterized by an H/H ratio of 0.49, while the B1 and Os2 cheeses were 0.63 and 0.60, respectively. In the present research, the B1 and Os2 cheeses were characterized with a higher HPI (0.50 and 0.47, respectively) than in the case of AW (0.41). After 1 and 2 months of storage, statistically favorable changes in the lipid indices were found (p < 0.05).

3.3. Physico-Chemical Properties of Cheese Samples

Acid-rennet cheeses made of goat’s milk were characterized by the same water activity value of 0.960 immediately after production (

Table 4. Mean values of water activity, pH value, oxidation-reduction potential and total acidity of acid-rennet goat’s cheeses after production and after 1 and 2 months of storage.

Parameter	Time [Month]			Cheese Symbol
	0	1	2
Water activity (aw)	0.960 Aa ± 0.006	0.947 Ba ± 0.005	0.935 Ba ± 0.002	AW
pH	5.88 Aa ± 0.01	6.10 Ba ± 0.01	5.81 Aa ± 0.08
TA [°SH]	30.00 Ba ± 1.41	29.50 Ba ± 1.12	40.25 Bb ± 4.82
ORP [mV]	313.75 Aa ± 7.46	414.25 Bb ± 3.27	447.15 Ca ± 12.88
Water activity (aw)	0.960 Aa ± 0.002	0.950 Ba ± 0.001	0.941 Ba ± 0.004	B1
pH	6.16 Bb ± 0.00	6.27 Ca ± 0.01	6.02 Ab ± 0.05
TA [°SH]	22.00 Aa ± 1.41	24.75 Aa ± 0.83	29.25 Ab ± 1.30
ORP [mV]	355.88 Ab ± 1.90	415.78 Bb ± 1.16	427.75 Ba ± 13.01
Water activity (aw)	0.960 Aa ± 0.006	0.940 Ba ± 0.001	0.932 Ba ± 0.003	Os2
pH	5.86 Ba ± 0.02	6.01 Ca ± 0.02	5.72 Aa ± 0.02
TA [°SH]	33.50 Aa ± 1.66	30.25 Ba ± 1.48	51.75 Ca ± 2.86
ORP [mV]	428.53 Bc ± 3.79	401.73 Aa ± 8.39	448.15 Ca ± 3.75

Explanatory: AW—farmer’s acid-rennet goat’s cheese with acid whey, B1—farmer’s acid-rennet goat’s cheese with L. brevis B1, Os2—farmer’s acid-rennet goat’s cheese with L. plantarum Os2; pH—pH value; ORP—oxidation-reduction potential; TA—titratable acidity. The values were expressed as means ± SD; means in the same row followed by different uppercase letters within the same sample at different times are significantly different (p < 0.05); means in the same column followed by different lowercase letters within samples at the same time are significantly different (p < 0.05) (n = 4).

). During storage, this value decreased significantly (p < 0.05) in the Os2, AW and B1 samples (to 0.932, 0.935 and 0.941, respectively), however, there were no statistical differences between them (p > 0.05).

The pH values of the cheeses after production were, respectively, for the control cheese AW, 5.88, for the B1 cheese, 6.16 and for the Os2 cheese, 5.86 (Table 4). In all the tests, after 1 month of storage, a significant (p < 0.05) increase in pH was observed and after 2 months of storage, the pH decreased again. Directly after production and after 2 months, the AW and Os2 cheeses did not differ from each other (p > 0.05).

Titratable acidity after production was 30.00°SH for the AW control sample, 22.00°SH for the B1 cheese and 33.50°SH for the Os2 cheese (Table 4). The B1 cheese showed a significant difference (p < 0.05). A decrease in the total acidity was observed in the case of the AW and Os2 cheeses after 1 month of storage, while in the case of the B1 cheese, there was an increase (not statistically significant, p > 0.05). On the other hand, after 2 months of storage, in the case of the AW and Os2 cheeses, a statistically significant (p < 0.05) increase in total acidity was observed.

In the tests of goat’s cheeses, an increase in the oxidation-reduction potential during storage was observed in all samples (Table 4). However, this increase was lower in the case of goat’s cheese samples with the addition of B1 and Os2 bacterial cultures in comparison to the control sample with the addition of whey (AW). A significant (p < 0.05) increase in ORP was observed in the control samples of AW and B1 after 1 month of storage. In the case of the Os2 cheese, a decrease in ORP was observed after 1 month of storage, followed by an increase after 2 months.

3.4. Sensory and Textural Evaluation of Acid-Rennet Goat’s Cheese

The results of the sensory evaluation of goat’s rennet cheese after production (month 0) and after 1 and 2 months of storage are presented in

Table 5. Results of sensory evaluation of goat’s acid-rennet cheeses after production and after 1 and 2 months of storage.

Parameter	Time [Month]			Cheese Symbol
	0	1	2
color	5.16 Aa ± 1.90	5.02 Aa ± 1.24	5.46 Aa ± 1.54	AW
milk’s fermentation o.	6.46 Ab ± 1.54	6.00 Aa ± 1.76	5.94 Aa ± 1.86
goat’s milk o.	6.11 Aab ± 1.65	6.00 Aa ± 1.87	5.99 Aab ± 1.67
fatty o.	3.82 ABa ± 1.29	3.12 Aa ± 1.45	4.93 Bb ± 1.82
sharp o.	4.22 Ba ± 1.83	4.11 Ba ± 1.73	3.67 Aa ± 1.92
mature cheese o.	4.84 Aa ± 1.76	4.98 Aa ± 1.68	5.39 Ba ± 1.50
softness	2.58 Aa ± 0.97	2.76 Aa ± 1. 79	3.37 Ba ± 1.16
moisture	7.53 Ba ± 1.25	7.55 Ba ± 1.95	5.09 Aa ± 1.93
elasticity	7.18 Ba ± 1.61	7.32 Ba ± 1.44	5.48 Aa ± 1.52
color	5.11 Ba ± 1.78	5.22 Ba ± 1.48	4.21 Aa ± 1.15	B1
milk’s fermentation o.	5.72 Aa ± 1.70	5.97 Aa ± 1.97	6.01 Aa ± 1.53
goat’s milk o.	6.84 Ab ± 1.23	6.01 Aa ± 1.84	6.67 Ab ± 1.53
fatty o.	3.79 Aa ± 1.82	3.45 Aa ± 1.97	3.76 Aa ± 1.74
sharp o.	4.46 Ba ± 0.98	4.66 Ba ± 1.09	3.53 Aa ± 1.65
mature cheese o.	4.92 Aa ± 1.73	4.12 Aa ± 1.37	4.78 Aa ± 1.24
softness	3.53 Ab ± 1.99	3.34 Aa ± 1.21	6.49 Bc ± 1.44
moisture	7.53 Aa ± 1.47	7.01 Aa ± 1.76	7.11 Ab ± 1.47
elasticity	7.38 Aa ± 1.14	7.77 Aa ± 1.93	7.49 Ab ± 1.35
color	4.36 Aa ± 1.73	4.64 Aa ± 1.22	4.49 Aa ± 1.29	Os2
milk’s fermentation o.	5.37 Aa ± 1.76	5.32 Aa ± 1.63	5.14 Aa ± 1.82
goat’s milk o.	5.46 Aa ± 1.82	5.33 Aa ± 1.25	5.72 Aa ± 1.98
fatty o.	3.66 Aa ± 1.76	3.23 Aa ± 1.89	5.39 Bb ± 1.71
sharp o.	4.23 Ba ± 1.97	4.00 ABa ± 1.79	3.44 Aa ± 1.80
mature cheese o.	4.70 Aa ± 2.63	4.97 Aa ± 1.57	4.97 Aa ± 1.49
softness	3.49 Ab ± 1.87	3.02 Aa ± 1.26	5.28 Bb ± 1.82
moisture	7.38 Ba ± 1.40	7.00 Ba ± 1.76	5.92 Aa ± 1.68
elasticity	6.89 Ba ± 1.68	6.99 Ba ± 1.71	5.37 Aa ± 1.32

Explanatory: AW—farmer’s acid-rennet goat’s cheese with acid whey, B1—farmer’s acid-rennet goat’s cheese with Levilactobacillus brevis B1, Os2—farmer’s acid-rennet goat’s cheese with Lactiplantibacillus plantarum Os2; o.—odor. The values were expressed as means ± SD; means in the same column followed by different lowercase letters within samples at the same time are significantly different (p < 0.05); means in the same row followed by different uppercase letters within the same sample at different times are significantly different (p < 0.05); (n = 16).

.

In the case of goat’s acid-rennet cheese B1, i.e., the cheese obtained by adding a B1 strain isolated from “bundz” cheese, the color changed after two months of storage—the color of the cheese became lighter. A different phenomenon was observed in the case of the Os2 sample and the sample of AW, where after two months of storage, the cheese was more of a creamy yellow than after the production.

The odor of lactic acid fermentation after two months of storage became less noticeable both in the case of the Os2 and AW cheeses (5.14 and 5.94, respectively). The opposite is the case for the B1 cheese (6.01), i.e., the odor was more intense. The lowest value of the milk’s fermentation odor corresponded to the Os2 cheese (p > 0.05). The intensity of the odor of goat’s milk, in the case of the AW cheese as well as Os2, was at a similar level of intensity. On the other hand, differences were noticed in the case of the B1 cheese sample, where the odor of the goat’s milk was most noticeable (p < 0.05) at the beginning, right after production (6.84). The intensity of the fatty odor in the Os2 cheese and AW cheese became more intense (p < 0.05) after two months of storage. Such a tendency was not observed in the case of sample B1. In contrast, for each sample, the pungent, irritating odor was less noticeable (p < 0.05) after two months of storage. On the other hand, the more intense odor of mature cheese was noticeable only in the case of the B1 sample. The time of ripening of the cheeses significantly (p < 0.05) changed their consistency to being softer, more elastic and less moist.

An instrumental examination of color is presented in

Table 6. Mean values of color parameters in cheese samples during storage.

Parameter	Time [Month]			Cheese Symbol
	0	1	2
L*	82.14 Aa ± 2.48	79.83 Ba ± 1.55	79.41 Ba ± 2.33	AW
a*	1.39 Aa ± 0.33	1.37 Aa ± 0.30	1.53 Aa ± 0.22
b*	8.71 Ac ± 0.58	9.40 Bc ± 0.36	8.49 Aa ± 0.38
L*	84.60 Ab ± 2.41	81.60 Ba ± 1.62	80.67 Ba ± 0.64	B1
a*	1.41 Aa ± 0.23	1.72 Bb ± 0.16	1.93 Cc ± 0.08
b*	8.16 Bb ± 0.60	7.46 Aa ± 0.51	7.56 Ab ± 0.33
L*	84.50 Bb ± 1.45	81.41 Bb ± 2.57	79.72 Aa ± 1.97	Os2
a*	1.97 Bb ± 0.22	1.90 Bc ± 0.30	1.80 Ab ± 0.18
b*	7.80 Aa ± 0.43	7.91 Ab ± 0.45	8.62 Ba ± 0.62

Explanatory: AW—farmer’s acid-rennet goat’s cheese with acid whey, B1—farmer’s acid-rennet goat’s cheese with L. brevis B1, Os2—farmer’s acid-rennet goat’s cheese with L. plantarum Os2; color parameters—L* indicates lightness, a* is the red/green coordinate and b* is the yellow/blue coordinate. The values were expressed as means ± SD; means in the same row followed by different uppercase letters represent significant differences (p < 0.05); (n = 20).

. Based on the tests performed, it was found that the test samples (B1 and Os2) were significantly (p > 0.05) brighter and less yellow than the control sample AW (L* values after production AW 82.14, B1 84.60, Os2 84.50). After storage, a gradual, statistically significant lightening of the cheeses was observed, with different changes of yellow and red colors, depending on the storage time. Both the sensory analysis and instrumental determination of color showed that sample B1 turned lighter after 2 months of storage, while sample AW turned more yellow.

The instrumental texture parameters of goat’s acid-rennet cheeses after production and after 1 and 2 months of maturation were presented in

Table 7. Texture instrumental parameters of goat’s acid-rennet cheeses after production and after 1 and 2 months of storage.

Parameter	Time [Month]			Cheese Symbol
	0	1	2
Hardness Cycle 1 [N]	93.50 Bb ± 18.62	68.80 Ab ± 4.43	102.67 Bc ± 14.18	AW
Adhesiveness [mJ]	1.02 Aa ± 0.86	1.13 Aa ± 0.99	1.18 Aa ± 0.76
Hardness Cycle 2 [N]	68.35 Ab ± 13.09	59.17 Ac ± 3.73	57.23 Ab ± 4.46
Cohesiveness	0.61 Ba ± 0.05	0.74 Cb ± 0.01	0.38 Aa ± 0.06
Springiness [mm]	8.87 Ba ± 0.07	8.67 Ba ± 0.14	8.11 Aa ± 0.56
Gumminess [N]	57.20 Bb ± 11.77	51.07 Bc ± 2.87	39.45 Ab ± 9.45
Chewiness [mJ]	506.83 Bb ± 101.56	442.37 Bc ± 20.15	324.06 Ab ± 91.61
Hardness Cycle 1 [N]	64.21 Aa ± 15.75	50.00 Aa ± 8.03	72.50 Ab ± 19.50	B1
Adhesiveness [mJ]	1.77 Aa ± 0.15	1.28 Aa ± 0.68	1.00 Aa ± 0.77
Hardness Cycle 2 [N]	45.30 Aa ± 12.07	43.30 Ab ± 6.79	56.42 Ab ± 14.54
Cohesiveness	0.61 Aa ± 0.13	0.75 Bb ± 0.01	0.64 ABb ± 0.06
Springiness [mm]	8.82 Ba ± 0.14	8.67 Ba ± 0.12	8.46 Aa ± 0.13
Gumminess [N]	38.72 Aa ± 10.61	37.45 Ab ± 5.94	46.26 Ab ± 12.32
Chewiness [mJ]	341.27 Aa ± 93.96	325.15 Ab ± 54.34	391.35 Ab ± 104.33
Hardness Cycle 1 [N]	51.54 Ba ± 4.93	42.94 Aa ± 4.63	46.38 ABa ± 5.77	Os2
Adhesiveness [mJ]	1.27 Aa ± 0.68	1.07 Aa ± 0.89	0.95 Aa ± 1.10
Hardness Cycle 2 [N]	35.78 Aa ± 4.27	35.85 Aa ± 3.10	31.49 Aa ± 2.53
Cohesiveness	0.57 Ba ± 0.08	0.71 Ca ± 0.01	0.37 Aa ± 0.09
Springiness [mm]	8.77 Ba ± 0.06	8.50 Ba ± 0.13	7.27 Aa ± 1.28
Gumminess [N]	29.50 Ba ± 4.73	30.52 Ba ± 2.84	17.09 Aa ± 3.01
Chewiness [mJ]	258.43 Ba ± 40.04	259.67 Ba ± 26.94	126.37 Aa ± 40.04

Explanatory: AW—farmer’s acid-rennet goat’s cheese with acid whey, B1—farmer’s acid-rennet goat’s cheese with L. brevis B1, Os2—farmer’s acid-rennet goat’s cheese with L. plantarum Os2. The values were expressed as means ± SD; means in the same column followed by different lowercase letters within samples at the same time are significantly different (p < 0.05); means in the same row followed by different uppercase letters within the same sample at different times are significantly different (p < 0.05); (n = 6).

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Based on the results, changes in the texture of acid-rennet goat’s cheese during the 2 months of storage can be noticed (Table 7). The values of hardness 1 and 2 were, significantly, the highest in the case of the AW cheese and statistically the lowest in the case of the Os2 cheese (93.50, 68.35, 51.54 and 35.78, respectively). During storage, hardness 1 and 2 decreased (in 1st month) and then increased (in 2nd month), regardless of the samples. The adhesiveness of samples B1 and Os2 were slightly lower during storage, however, there were not any statistical differences between the samples (p < 0.05). The cohesiveness of the acid-rennet cheeses during storage firstly increased in the 1st month and then decreased in the 2nd month, regardless of the samples. In the case of each sample in the 2nd month of storage, the springiness decreased significantly. Both the gumminess and chewiness, in the case of the Os2 and AW samples, decreased and were statistically lower after 2 months.

4. Discussion

Goat’s milk is a very important food product in many regions of the world and the interest in the production of goat’s milk products has increased over the past decade [45,46]. Goat’s milk is also an excellent material for the development of such functional foods as cheeses [47,48]. In terms of suitability for processing, goat’s milk differs significantly from cow’s milk. Due to the lower content of casein and its smaller share in the total amount of proteins, the cheese yield is lower, and the curd obtained is more delicate and less firm [49]. Scientific interest in artisanal cheese is growing because it represents a source of environmental bacteria with specific, potential health benefits [50].

In the present work, the acid-rennet cheeses from organic goat’s milk fermented with L. brevis B1 and L. plantarum Os2, or acid whey as a control, were obtained under industrial conditions and evaluated. According to FAO/WHO General Standard for Cheese [51], the produced cheeses can be classified as semi-hard or partially skimmed. The examples of semi-hard cheeses are Colby and Monterey (stirred-curd Cheddar-type cheeses), a number of British Territorial varieties (Caerphilly, Lancashire and Wensleydale) and cheeses such as Majorero (Spain) or Bryndza (Slovakia) and Bundz (Poland) [52]. The tested cheese samples were mostly similar to the Bundz type and prepared according to the producer recipe, replacing the acid whey with a dedicated starter culture B1 or Os2. It was found that lactic acid bacteria (LAB) were the predominant microbiota (about 8 log CFU/g) during cheese storage and the use of the B1 and Os2 culture and did not affect the microbiological quality of the cheeses. However, in case there was no confirmation of colonies of presumptive LAB on the medium (MRS agar), it was taken into consideration that other specific microorganisms could also grow, i.e., Staphylococcus sp. Essentially harmful are S. aureus enterotoxins producers and they should be controlled during the production process. When the detected value is >10⁵ CFU/g, the cheese batch must be tested for staphylococcal enterotoxins [53]. It should also be emphasized that the cheese samples were made from unpasteurized, raw goat’s milk, which was not free of native microbiota. However, even in the control sample, the acid whey was added to control the fermentation process. The endogenous microbiota can grow in the milk during fermentation, next to the starter cultures; however, carefully selected starter cultures of bacteria strains are able to dominate during the milk’s fermentation [54]. Lactobacilli (predominantly Lactobacillus brevis, Lactobacillus plantarum, Lactobacillus paracasei and Lactobacillus casei) constitute the majority of nonstarter lactic acid bacteria in Cheddar, creamy, and some artisanal cheeses. Typical counts of Lactobacillus cells range from 10 to about 10⁸ CFU/g of the cheese samples [55,56,57]. In comparison, Mushtaq et al. [58] used L. brevis as a probiotic starter culture and found that counts of LAB during 30 days of storage period were approximately 9–6 log CFU/g. Although, in our study, we do not provide the identification of the LAB, the changes in the chemical and physical properties, as well as the texture and sensory characteristics, were observed in samples with L. brevis B1 and L. plantarum Os2, in comparison to the AW control. These observations gave an initial proof of domination of the selected starter cultures and could be a premise for further research.

We have found that the level of Enterobacteriaceae in the final product was very high (5.57–6.66 log CFU/g), which suggests the need to improve the hygienic condition of the process. The important observation is that the presence of the Enterobacteriaceae family was successfully reduced during storage. On the other hand, the samples were free of Listeria monocytogenes and Salmonella spp., which was in line with the UE regulation [53], as well as the studies by Dauber et al. [9], Andretta et al. [59], de Medeiros Carvalho et. al. [60] and De la Rosa-Alcaraz et al. [50]. Moreover, the selected bacterial strains L. brevis B1 and L. plantarum Os2 possess strong antibacterial properties [19,20]. The isolation of the LAB strains with anti-microbiological, mainly anti-listerial activity from artisanal cheese samples is quite common [16], which could be the reason of the phenomenon.

Microbial dynamics in cheese are affected by several factors, such as milk composition, salting, starter culture, rennet addition, temperature, relative humidity (85–97%), pH, redox potential, water activity and moisture [61]. Among the several factors that influence the physicochemical properties of cheese, the acidity is crucial as it directly affects the stability of casein micelles and milk minerals [62]. In our study, the pH value was in the range of 5–6, which is the typical value for most cheese varieties [52], however, it was lower than in study of Guimarães et al. [63]. The differences in the pH values could be caused by the different composition of the starter cultures and their interaction. After 1 month of storage, a significant (p < 0.05) increase in pH was observed, which may have been caused by the utilization of lactic acid, the formation of a non-acid decomposition product and the release of alkaline protein decomposition products [64,65]. Both the starter bacteria used, L. plantarum Os2 and L. brevis B1, are heterofermentative LABs that use the phosphoketolase pathway to produce a mixture of lactic acid, ethanol, acetic acid and CO₂ as products of hexose fermentation [55]. These bacterial strains represented a similar slight acidification model, however, the Os2 strain seems to be more effective as the TA value after 2 months of storage was the highest in the samples of cheeses fermented with the L. plantarum Os2 strain. In addition, the oxidation-reduction potential increased during storage in all samples and was higher than in the study by Abraham et al. [66] where cheeses such as Camembert, Cheddar and Comté reached an ORP between 100 and 350 mV. Some authors [67,68,69] claim that LAB strains show the antioxidant activity, which is why the high value of the oxidation-reduction potential can be explained by the high microbiological activity of the LAB strains and their enzymatic capacity. In the tested cheeses, no additional substances were used to influence the oxidation processes and the high values of the redox potential testify to the superiority of the oxidation reaction processes. The oxidation-reduction potential modifications can affect both the microbiological quality (biological activity of non-starter lactic acid bacteria) and the sensorial properties (synthesis and/or stability of aroma compounds) of cheeses [70].

The biochemical changes that occur during cheese maturation can be divided into primary metabolic changes such as glycolysis, lipolysis and proteolysis, followed by secondary biochemical changes such as fatty acid and amino acid metabolism, which play an important role in the production of secondary metabolites, including compounds responsible for the flavor bouquet [62]. The composition of cheese samples prepared in this study was typical and comparable to others [50,70,71,72].

Goat’s milk contains slightly less lactose than cow’s milk (goat’s milk 4.1–4.5% of lactose, cow’s milk 4.6–4.7% of lactose). Most of the lactose is found in whey, which is the liquid that is separated from the solid cheese curd during the cheese-making process. The longer the cheese has been aged, the less lactose will remain in the final product [73]. In our study, no lactose was found in the cheese samples. During the maturation process, protein and fat are continuously broken down. Casein is first hydrolyzed to long chain peptides which are then broken down into short chain peptides. Some of the casein eventually breaks down into amino acids and volatiles. When dispersed into small fat globules, the large fat globules are further broken down into ketones, aldehydes and lactones, which break down into volatile substances and free fatty acids [74]. In the present study, the main fatty acids present in the tested acid-rennet cheeses were palmitic C16:0, oleic C18:1cis9, stearic C18:0 and myristic C14:0, which were also observed in the studies by Popović-Vranješ et al. [75]. It should also be underlined that there was a significant increase in omega-3 and in the cholesterol content in all samples after storage was denoted. Similar results were obtained by Burgos et al. [76]. On the other hand, much higher values of the cholesterol content were obtained in the studies by Barłowska et al. [77], where 358–370 mg/100 g of fat were denoted. The results of the SFA, MUFA and PUFA content obtained in our work differ from the results presented by Paszczyk and Łuczyńska [32], where the sum of the saturated acids in the goat’s cheeses was 58.08% on average, with the advantage for unsaturated acids MUFA and PUFA (23.66 and 3.49%, respectively). In turn, higher SFA values were observed in the studies by Schettino-Bermúdez et al. [78]. The high levels of SFA in milk naturally occur and dairy products are often correlated with the adverse effects of these foods and with the development of several civilization diseases, including cardiovascular disease, type 2 diabetes, obesity and cancer [79]. The differences in the chemical composition between the cited studies of the cheeses may have resulted from the different production and maturation conditions of the goat’s cheeses, as well as the starter culture used for their production. Due to the observed changes in the fatty acid profile and in the microstructure of the goat’s cheeses during the ripening period in the present study, it is recommended to mature the goat’s cheese for at least 30 days before consumption or sale.

To assess the health properties of evaluated acid-rennet cheeses the Lipid Quality Indices were calculated. The Index of Atherogenicity (AI) shows the relationship between the sum of saturated fatty acids, which are considered pro-atherogenic and the sum of unsaturated fatty acids [33]. The AI index values in tested cheese samples were at an average level (2.06–2.50) in all the samples, which is a rather small value, due to the AI value ranging from 1.42 to 5.13 for dairy products [33]. The Index of Thrombogenicity characterizes the thrombogenic potential of fatty acids, indicating the tendency to form clots in blood vessels. The lower TI characterizes the healthier the product for the cardiac system [33].

Interestingly, in our study a favorable share of DFA compared to OFA in B1 and Os2 goat’s cheeses was observed. On the other hand, control AW goat’s cheese was characterized by a higher OFA index. In the studies by Paszczyk and Łuczyńska [32], higher OFA values were observed in goat’s cheeses compared to DFA, as well as a lower AI value and a higher TI value. The hypocholesterolemic effect is mainly to reduce the absorption of cholesterol from the gastrointestinal tract [34]. The H/H ratio characterizes the relationship between the hypocholesterolemic fatty acids and hypercholesterolemic fatty acids and may more accurately reflect the effect of the fatty acids’ composition on cardiovascular diseases. Additionally, the health-promoting index (HPI), which was proposed to assess the nutritional value of dietary fat and focuses on the effect of fatty acids composition on CVD (Cardiovascular Disease) was found to be higher for B1 and Os2 goat’s cheeses. High HPI dairy products are believed to be more beneficial to human health [33]. To sum up, the tested cheeses with the addition of L. brevis B1 and L. plantarum Os2 bacterial cultures were characterized by more favorable Lipid Quality Indices compared to the control with the addition of acid whey.

The sensory quality of foods is one of the main factors influencing the acceptance of a product on the market. For this reason, studying factors that influence the sensory quality are crucial in food product development. In the presented study, it was shown that some odor attributes, especially those with a negative sensory influence were of importance for the overall sensory characteristics. It is well known that even a slight increase in the intensity of negative attributes, mainly flavor or odor, are connected with a severe decrease of the overall sensory quality of food products [80,81,82]. In the present study, a negative odor intensity (sharp) was, on average, in the range of 3.44–4.66 c.u. and was not different between samples. Furthermore, the unpleasant odor typical for goat’s milk was most noticeable at the beginning and decreased during the samples’ storage. It could be due to the LAB metabolic activity, for example, the production of carboxylic acids in cheese due to their lipolytic activity. It was found that the typical, pleasant flavor of milky goat’s cheese was related to the free fatty acids, especially free hexanoic acid, octanoic acid and n-decanoic acid [62]. Similarly, Calvo et al. [83] found that during the storage of ripened goat’s cheese, as the external appearance features deteriorated and the intensity of the “goat” flavor decreased, it became less noticeable. Other attributes were not differentiated between the tested cheese samples. Due to the COVID-19 pandemic, oral evaluation was omitted, so the overall sensory quality was not assessed. However, it can be concluded that the odor of the tested cheese samples was similar in all samples.

Color and texture are important criteria for assessing the quality of a cheese, as these two parameters are important for consumers when making a decision to buy a product [84]. The color is one of the most important quality attributes, noticed at first glance, on the basis of which the consumer assesses the acceptability of the product [83]. The results of present study showed that the L* value decreases significantly, whereas the a* and b* values were found to remain unchanged in all the samples during storage, which is in agreement with other authors studies [58,85]. The texture characteristics become of significant importance when there are no flavor defects in the samples [44]. It must be considered by a production step that even the high quality of the texture attributes will not ensure the proper acceptance of the product. In the present study, we observed that in the 1st month of storage, the hardness decreased and then in the 2nd month, it increased regardless of the cheese samples. In turn, the cohesiveness of the acid-rennet cheeses during storage firstly increased and then decreased regardless of the samples. Similar trends were observed in the study of Zaravela et al. [73]. In the study of Jia et al. [62], the hardness of the semi-hard goat’s cheese samples showed an overall upward trend with the increasing maturity and the cohesiveness decreased slightly as the maturation time progressed. This phenomenon is a consequence of protein and fat hydrolyzation during the maturation process.

During the 2 months of storage of the goat’s cheese samples, the changes of the instrumentally evaluated texture were noted. The hardness increased regardless of the samples and was the highest in the case of the AW cheese. At the same time, the springiness, gumminess and chewiness in the case of samples Os2 and AW decreased. In the presented study, the sensory analysis of the cheeses’ moisture was in agreement with the instrumental measurement of water activity and water content. Gámbaro et al. [71] showed that the moisture content of fresh cheese (48.7%) was higher than that of ripened cheese (42.7%), so the storage time influences the changes in cheese moisture. When comparing the sensory analysis of the texture and instrumental texture determination, there were some discrepancies. According to the evaluators, the cheese, after 2 month of storage, became softer and more elastic. In turn, TPA analysis shows that after 2 months of storage, the hardness increased and springiness decreased regardless of the sample, similar to the García et al. [74] study. To sum up, sample B1 turned out to be more soft and elastic according to the sensory analysis, as well as being less hard and springy in the instrumental texture analysis.

5. Conclusions

When summarizing the obtained research results, it should be stated that the technology of acid-rennet curd production with the use of selected, environmental bacterial cultures is in line with the current trends related to the innovation of fermented food development with high nutritional quality. Scientific studies have shown that a daily diet enrichment with 10⁶–10⁹ CFU/mL of beneficial lactic acid bacteria cells after just a few weeks may increase the number of natural killer cells in the blood serum, and increase the activity of macrophages and lymphocytes. Moreover, the immunomodulatory action of lactic acid bacteria may further reduce allergic reactions in humans.

According to our study it can be concluded that the selected cultures of L. brevis B1 and L. plantarum Os2 isolated from traditional cheeses from a Polish mountain can be applied to goat’s milk cheese production. Bacteria cultures affected the microbiological, physical, chemical and sensory quality of the cheese samples. The lactic acid bacteria count in the cheese samples was at a high level of about 8 log CFU/g. Moreover, the cheeses with the addition of L. brevis B1 and L. plantarum Os2 bacterial cultures were characterized by more favorable Lipid Quality Indices than for the control with the addition of acid whey. The produced cheeses were at a similar sensory quality and after storage, the experimental cheeses (B1 and Os2) were more elastic and softer than the control sample (AW).

However, it should be underlined that after taking into account high number of the Enterobacteriaceae bacteria group, as well as high the pH and water activity values in the samples, the cheeses produced in this way cannot be recommended for consumption. Therefore, future studies should focus on increasing the microbiological quality and the deeper analysis of the cheese ripening processes, including the lipolysis and proteolysis rates. The next step could also be the identification of culture strains from food samples to provide proof of the potential probiotic properties of goat’s cheeses. Such innovative products can be excellent carriers of environmental bacteria with potentially probiotic properties and can be widely used for the enrichment of the intestinal microbiota of humans, especially for the Polish population.