Enhancing the Biopreservative Effect of Non-Starter Lactic Acid Bacteria Using Soluble Fiber During Cheese Ripening

Abstract

Cheddar cheese ripening creates favorable conditions for desired microbial changes but also allows survival and outgrowth of spores like Clostridium tyrobutyricum and Bacillus licheniformis, leading to late-blowing defects. In the first phase of the study, NSLAB dynamics were evaluated in the presence of spores, where pilot-scale cheeses (110 L) were produced in four treatments: control, T1 (BL), T2 (CT), and T3 (BL+CT), each inoculated at 2.0 Log₁₀ CFU/mL with spores. Results showed that SLAB declined from 8.0 to 0.2 Log₁₀ CFU/g, while NSLAB increased from 2.0 to 8.5 Log₁₀ CFU/g by the third month and remained stable thereafter. Spore counts reached 2.94 ± 0.02 (T2) and 2.48 ± 0.03 (T3) Log₁₀ CFU/g, with visible spoilage signs appearing after five months, indicating that native NSLAB populations were inadequate to control late-blowing defects. In this study, the effect of soluble fiber (inulin) in stimulating NSLAB was evaluated by incorporating 1% inulin into Cheddar cheese across four treatments: T1 (C SF), T2 (BL SF), T3 (CT SF), and T4 (BL+CT SF). Inulin addition resulted in significantly higher NSLAB counts (>10.5 Log₁₀ CFU/g) and suppressed spore levels (<0.91 ± 0.03 Log₁₀ CFU/g), with no spoilage observed. Inulin addition selectively enhanced beneficial NSLAB, suppressing spore-forming bacteria and preventing late-blowing defects without affecting cheese quality. This provides a natural, sustainable strategy to enhance microbial safety during Cheddar cheese ripening.

1. Introduction

Cheese is a complex, fermented dairy product characterized by a wide variety of textures, flavors, and aromas, shaped by milk composition, microbial activity, and ripening conditions. Ripening is a biochemical and microbiological transformation process that lasts from weeks to months, during which microbial enzymes and metabolic byproducts drive the breakdown of proteins, fats, and residual lactose, generating the sensory qualities of the final product [1,2,3]. While many of these changes are desirable, the ripening environment can also favor the survival and growth of spoilage microorganisms, particularly spore-forming bacteria such as Clostridium and Bacillus species [4]. These spores, introduced via raw milk, poor hygiene, or environmental sources, are highly resistant to heat and desiccation, enabling them to survive pasteurization and persist through cheese manufacture [5]. Under favorable conditions, reduced redox potential, low oxygen, and specific nutrients, they can germinate during ripening, causing defects. The outgrowth of spore-formers, particularly Clostridium tyrobutyricum, is associated with butyric acid fermentation, gas production, off-flavors, and the late-blowing defect in semi-hard and hard cheeses, which also disrupts normal microbial succession and flavor development [6,7]. Traditional approaches to control late-blowing, including adjunct cultures [8], high-pressure processing [9], addition of sodium nitrate [10], microfiltration and bactofugation [11], bacteriophages, nisin, and lysozyme [12], have shown limited efficacy in semi-hard and hard cheeses. Despite these efforts, the persistent survival of spores highlights a technological gap in achieving consistent microbial control through natural, clean-label means.

A novel approach is the use of prebiotics, defined as substrates selectively utilized by host microorganisms, conferring health benefits [13]. Among prebiotics, inulin, a fructan composed of β-(2→1) linked fructose units terminating in a glucose residue, is the most widely studied, followed by galactooligosaccharides (GOS) and fructooligosaccharides (FOS) [14]. Soluble fibers like inulin and FOS are water-soluble and fermentable, enabling them to act as selective growth substrates for beneficial microorganisms. In dairy systems, these fibers have been shown to promote lactic acid bacteria growth, enhance functional properties, and improve texture and moisture retention [15,16,17]. However, previous studies have primarily focused on the nutritional or functional benefits of inulin rather than its role in microbial ecology or spoilage suppression during cheese ripening. The potential of inulin to act as a natural biopreservative by stimulating non-starter lactic acid bacteria (NSLAB), key contributors to flavor and microbial stability, has not been systematically explored. So, this study investigates the targeted use of inulin as a biopreservative agent in Cheddar cheese to selectively enhance NSLAB activity and inhibit spore-forming spoilage organisms such as Clostridium and Bacillus. Inulin was specifically chosen because of its well-documented selectivity toward Lacticaseibacillus and Lactobacillus species [13,18], its compatibility with dairy matrices, and its ability to promote metabolic pathways that generate inhibitory compounds such as organic acids and bacteriocins [19].

Therefore, this research addresses a critical gap by linking the prebiotic function of inulin with microbial safety enhancement in ripened cheese. By examining how inulin modulates NSLAB populations and suppresses spore outgrowth, this study proposes a sustainable, natural strategy to prevent late-blowing defects and improve the quality and shelf stability of Cheddar cheese.

2. Materials and Methods

2.1. Experimental Design

Each cheese was made in batches of 110 kg milk at Davis Dairy Plant (DDP) at South Dakota State University, Brookings, and two independent trials were conducted for each of the following: experimental control with soluble fiber, T1 (C SF); cheese spiked with aerobic spore-former Bacillus licheniformis (BL) at 2log₁₀ CFU/g and soluble fiber, T2 (BL SF); cheese spiked with anaerobic spore-former Clostridium tyrobutyricum (CT) at 2log₁₀ CFU/g and soluble fiber, T3 (CT SF); and a mixture of both and soluble fiber, T4 (BL+CT SF). Cheese samples were stored in the ripening room at 7 °C for six months, and microbial and chemical analyses were performed monthly in triplicate.

2.2. Materials

Pasteurized standardized milk was procured from Davis Dairy Plant, SDSU, Brookings, SD, USA. The starter culture for the cheese manufacturing was procured from CHR Hansen (DVS 970, 500U, Milwaukee, WI, USA). The aerobic spore-former (Bacillus licheniformis 6634) and the anaerobic spore-former (Clostridium tyrobutyricum VPI 5392) isolates were procured from ATCC (Manassas, VA, USA). Soluble Fiber (inulin) was procured from Natural Taste International Inc., San Diego, CA, USA.

2.3. Spore Preparation

2.3.1. Aerobic Spores

Spores of B. licheniformis (6634, ATCC, USA) were prepared by spreading 1 mL of actively growing broth culture of B. licheniformis onto TSA plates and incubated under aerobic conditions at 37 °C for up to 3 days to promote sporulation, and spores were harvested by flooding the plate surface with 10 mL of sterile distilled water, allowing it to soak for 2 to 3 min, and then gently scraping with a sterile spreader. The spore suspension was collected into sterile 50 mL centrifuge tubes and centrifuged at 4500× g for 30 min. The resulting pellets were washed twice by resuspending in 20 mL sterile distilled water, followed by centrifugation at 4500× g for 30 min at 20 °C. The final spore pellets were resuspended in 10 to 15 mL of sterile distilled water and heat-treated at 80 °C for 20 min to eliminate any remaining vegetative cells [20].

2.3.2. Anaerobic Spores

Spores of C. tyrobutyricum (VPI 5392, ATCC, USA) were prepared by spreading 1 mL of actively growing broth culture of Cl. tyrobutyricum onto RCA plates and incubated under anaerobic conditions at 37 °C for up to 7 days to promote sporulation, and spores were harvested by flooding the plate surface with 10 mL of sterile distilled water, allowing it to soak for 2 to 3 min, and then gently scraping with a sterile spreader. The spore suspension was collected into sterile 50 mL centrifuge tubes and then centrifuged at 4500× g for 30 min. The resulting pellets were washed twice by resuspending in 20 mL sterile distilled water, followed by centrifugation at 4500× g for 30 min at 20 °C. The final spore pellets were resuspended in 10 to 15 mL of sterile distilled water and heat-treated at 80 °C for 20 min to eliminate any remaining vegetative cells [20].

2.4. Pilot-Scale Manufacturing of Cheddar Cheese with Added Soluble Fiber (Inulin)

For Cheese manufacturing, pasteurized and standardized whole milk was obtained from the Davis Dairy Plant (SDSU, Brookings). The cheese manufacturing process was done in a Double O cheese vat (Kusel Equipment Co., Watertown, WI, USA). After adjusting the milk temperature to 32 °C, mesophilic starter culture (DVS 970, CHR Hansen, USA) and 1% (w/v) inulin (Natural Taste International Inc., San Diego, CA, USA) was added to the milk. After a 0.2 decrease in the pH, rennet was then added and allowed to set for 30 to 35 min. The setting of the curd was checked with a stainless-steel rod by inserting it vertically and lifting it with an inclination to check if any curd particles were sticking or not. After that, the curd was cut horizontally and vertically, respectively, with the cheese knives, and allowed to rest for 5 min. Then, the curd was cooked slowly to 39 °C at a rate of 1 °C increase after every five min. After cooking, the whey was drained through drainage with a strainer to prevent curd loss. The curd then went through the process of cheddaring (packing, cutting, piling, re-piling) until the pH reached 5.5. After that, the curd was cut into small cubes (2.5 cm × 2.5 cm), and salt (non-iodized) (Great Value, Brookings, SD, USA) was added by sprinkling over the curd (dry salting) and mixed properly. The curd was then filled into hoops and pressed at 15 Pa for 16 h at room temperature. The pressed curd was then vacuum packaged in a 6 × 6 × 6 inches block and kept at 7 °C for 6 months for ripening.

2.5. Microbial Analysis

2.5.1. Total Plate Counts (TPC) and Spore Counts (SC) of Pasteurized Milk

Microbial analysis of pasteurized milk was performed using the standard pour plate method [21]; total plate counts were enumerated on Tryptic Soy Agar (TSA) incubated at 37 °C for 48 h, and spore counts were enumerated on tryptic soy agar after heat treatment of milk at 80 °C for 20 min. to eliminate any vegetative cells and incubated at 37 °C for 24–48 h.

2.5.2. Microbial Quality of Soluble Fiber (Inulin)

Microbial analysis of inulin was done using the standard pour plate method as total plate counts were enumerated on Tryptic Soy Agar (TSA) incubated at 37 °C for 48 h and spore counts were enumerated on tryptic soy agar after heat treatment of mixture of inulin (10 g) and distilled water (90 mL) at 80 °C for 20 min. to eliminate any vegetative cells and incubated at 37 °C for 48 h. Coliform counts in the inulin powder were determined using the pour plate method on violet-red bile Agar (VRBA) incubated at 35 °C for 48 h.

2.5.3. Sample Preparation of Cheese for Microbial Enumeration

For each analysis, 11 g of cheese was aseptically transferred into 99 mL of 2% (w/v) sodium citrate buffer to ensure proper dispersion of microbial cells. The mixture was homogenized in a high-speed stomacher (Stomacher 400 Circulator, Seward, Welwyn Garden City, Hertfordshire, UK) for 3 min. Serial dilutions were prepared using sterile PBS (Fisher Scientific, Geel, Belgium), and appropriate dilutions were plated on selective and non-selective media for microbial enumeration [21].

2.5.4. Total Plate Counts of Cheese Samples with Added Inulin

Following homogenization, serial dilutions of the cheese samples were prepared using sterile PBS (Fisher Scientific, Geel, Belgium). For the enumeration of total plate counts, the standard pour plate method [22] was used, where appropriate dilutions were pour-plated in triplicate on TSA (Remel, ThermoFisher, Lenexa, KS, USA) using a sterile pipette. The plates were incubated aerobically at 37 °C for 48 h. After incubation, colonies were counted manually using a standard colony counter, and results were expressed as colony-forming units per gram (CFU/g) of cheese. All samples were analyzed in triplicate, and the mean values were recorded and converted to log₁₀ CFU/g for statistical analysis.

2.5.5. Spore Counts of Cheese Samples with Added Inulin

For spore enumeration, aliquots of the homogenized and diluted samples were subjected to heat treatment at 80 °C for 20 min in a shaking water bath (BS-06, JeioTech, Daejeon, Republic of Korea) to inactivate vegetative cells, ensuring that only heat-resistant spores were quantified [23]. Following heat treatment, samples were immediately cooled in an ice bath and plated in triplicate on selective media. Aerobic spore-forming bacteria were enumerated on TSA (Remel, ThermoFisher, KS, USA) and incubated aerobically at 37 °C for 48 h. Anaerobic spore-forming bacteria were enumerated on RCM agar (OXOID, Hants, Basingstoke, Hampshire, UK) and incubated anaerobically at 37 °C for 48 h using anaerobic jars with anaerobe container system sachets with the indicator (BD, Cockeysville, MD, USA). Colonies were manually counted and reported as colony-forming units per gram (CFU/g) of cheese. All counts were performed in triplicate and expressed as log₁₀ CFU/g (Limit of Detection—1 Log). Strict aseptic techniques were followed throughout the procedure to ensure the accuracy and reproducibility of the results.

2.5.6. Starter and Non-Starter Lactic Acid Bacteria (NSLAB) Counts of Cheese Samples with Added Inulin

After homogenization of 11 g of cheese in 99 mL of 2% sodium citrate buffer using a high-speed stomacher (Stomacher 400 Circulator, Seward, Welwyn Garden City, Hertfordshire, UK) for 3 min, serial dilutions were prepared in sterile PBS (Fisher Scientific, Geel, Belgium). For starter Lactococci, appropriate dilutions were plated on M17 agar (OXOID, Hants, UK), and for NSLAB, serial dilutions were plated on De Man, Rogosa, and Sharpe (MRS) agar (BD Difco, Sparks, MD, USA) in triplicate. M17 plates were incubated aerobically at 37 °C for 72 h, and MRS plates were incubated anaerobically at 37 °C for 72 h using anaerobic jars with anaerobe container system sachets with an indicator (BD, MD, USA) to ensure optimal growth conditions for obligate and facultative anaerobes [8,24]. After incubation, colonies were counted manually and expressed as colony-forming units per gram (CFU/g) of cheese. All analyses were performed in triplicate, and the results are reported as mean log₁₀ CFU/g.

2.5.7. Identification of NSLAB Isolates Using MALDI-TOF

Representative colonies from MRS plates were randomly selected based on distinct morphological characteristics and subcultured on their respective media to obtain pure isolates. Species-level identification of isolates was performed using Matrix-Assisted Laser Desorption/Ionization–Time of Flight (MALDI-TOF) at Animal Disease Research and Diagnostic Laboratory (ADRDL), South Dakota State University (SDSU), Brookings, SD, USA.

2.6. Physicochemical Analysis

2.6.1. Moisture Content

The moisture content of cheddar cheese was determined by [25], according to which 3 g of the cheese sample was grated on empty dried aluminum dishes and kept in a hot air oven (Isotemp Oven, Fisher Scientific, Waltham, MA, USA) at 102 ± 2 °C for 5 h, and then the dishes were cooled to room temperature in a desiccator, and the dried weight was measured. The moisture content of the cheese sample was determined using Equation (1).

$$\mathrm{Moisture} \%.={\displaystyle \frac{(W1-W3 )\times 100}{W1-W2}}$$

(1)

where

W3—the weight of the dish with the dried sample (g);

W2—the weight of the empty dish (g);

W1—the weight of the dish with the sample (g).

2.6.2. Protein Content

The total protein content of cheese samples was determined by the micro Kjeldahl method [26] based on the determination of organic nitrogen concentration. A 0.3 to 0.5 g sample of cheese was placed into Kjeldahl tubes, and 10 mL of H₂SO₄ (98%, ACS Grade, VWR Chemicals, Radnor, PA, USA) was added along with catalyst tablets (FOSS Kjeltec, Runcorn, Cheshire, UK). The samples were then digested in a digester (DKL heating Digester, Velp Scientifica, NY, USA) at a range of 350–400 °C for 4 h to convert nitrogen into NH₄⁺. Then, the samples were distilled into a distillation unit (UDK 129, Velp Scientifica), and the nitrogen was trapped in saturated boric acid (ACS, Spectrum Chemicals, Gardena, CA, USA) with the mixed indicator (Methyl red and Bromocresol green). The boric acid with dissolved nitrogen was then titrated with 0.1 N hydrochloric acid (HCl) till the green color changed to light pink. The total Nitrogen content of the cheese samples was determined using Equation (2), and protein content was determined using Equation (3).

$$\mathrm{Nitrogen} (\mathrm{N}) \%={\displaystyle \frac{1.4007\times N\times (Vs-Vb)\times 100}{W}}$$

(2)

where

N—normality of HCl;

V_s—volume of HCl used for sample (mL);

V_b—volume of HCl used for blank (mL);

W—weight of sample (g).

$$\mathrm{Protein} \%=N\%\times 6.38$$

(3)

2.6.3. Fat Content

The fat content of cheese was determined using the Modified Mojonnier Ether Extraction method [27]. The fat content of the cheese samples was determined using Equation (4).

$$\mathrm{Fat}\%={\displaystyle \frac{W3-W2\times 100}{W}}$$

(4)

W3—the weight of the dish with the dried sample (g);

W2—the weight of the empty dish (g);

W—the weight of the sample (g).

2.6.4. pH

Cheese pH was determined by homogenizing 20 g of grated cheese with 12 mL of deionized water, followed by measurement using a calibrated pH meter (Orion Lab Star PH111, Thermo Scientific, Waltham, MA, USA) [28].

2.6.5. Free Fatty Acids

Free fatty acids of the cheese samples were determined by the method explained by Asif et al., 2023, according to which 50 g of sample was mixed with absolute and neutralized ethanol with 0.1 N NaOH and then titrated with NaOH, and FFA was calculated in terms of oleic acid (Equation (5)) [29].

$$\mathrm{Free} \mathrm{Fatty} \mathrm{Acid} (\%\mathrm{oleic} \mathrm{acid})={\displaystyle \frac{V\times N\times 28.2}{W}}$$

(5)

where

V—volume of NaOH used for titration (in mL);

N—normality of NaOH solution;

W—weight of the sample (in grams);

28.2—milliequivalent weight of oleic acid.

2.6.6. Visual Inspection of Cheese Samples for Bloating/Slits/Holes

During ripening, cheese samples were routinely inspected for visual and olfactory changes to monitor gas formation and the presence of butyric acid. Cheeses exhibiting signs of blowing were identified by gas accumulation within the packaging, the development of irregular eyes, cracks, and splits in the cheese matrix, and the rancid odor characteristic of butyric acid production.

2.7. Statistical Analyses

All data were analyzed using two-way analysis of variance (ANOVA) to determine the effects of ripening time, treatment, and their interaction on the measured parameters at p < 0.05. Time and treatment were considered fixed factors, and where significant differences were found, mean comparisons were conducted using Tukey’s post hoc analysis. The statistical analyses were performed using OriginPro 2025 (OriginLab Corporation, Northampton, MA, USA). Results are presented as means ± standard deviations of triplicate measurements unless otherwise stated. The data for the non-soluble fiber cheese sample was adopted from the previous part of the study by Kaushik et al., 2025 [30].

3. Results

3.1. Microbial Analysis

3.1.1. Microbial Quality of Pasteurized Milk Used for Cheese Manufacturing

All four milk batches showed consistently low total plate counts (TPC), ranging from 2.55 to 2.64 Log₁₀ CFU/mL, and no detectable spore-formers were found (

Table 1. Total plate counts (Log₁₀ CFU/mL) and spore counts (CFU/mL) of different batches of milk used for different cheese manufacturing with soluble fiber.

Pasteurized Milk	TPC (Log10 CFU/mL)	Spore (Log10 CFU/mL)	pH
Batch 1	2.55 ± 0.07	BDL	6.58 ± 0.02
Batch 2	2.55 ± 0.07	BDL	6.58 ± 0.02
Batch 3	2.64 ± 0.06	BDL	6.56 ± 0.01
Batch 4	2.64 ± 0.06	BDL	6.56 ± 0.01

All values are presented as mean ± standard deviation; differences were not statistically significant (p > 0.05). Below detection limit (BDL)—spore counts < 1 Log₁₀ CFU/mL; Batch 1—Milk used for manufacturing of control cheese with soluble fiber; Batch 2—Milk used for manufacturing cheese by incorporating aerobic spores (Bacillus licheniformis) with soluble fiber; Batch 3—Milk used for manufacturing cheese by incorporating anaerobic spores (Clostridium tyrobutyricum) with soluble fiber; Batch 4—Milk used for manufacturing cheese by incorporating aerobic and anaerobic spores (Bacillus licheniformis & Clostridium tyrobutyricum) with soluble fiber.

). This confirms that the milk used for cheese production with soluble fiber was microbiologically safe and of high quality, aligning well with the previous baseline trials without fiber, where TPC also remained under 3 Log₁₀ CFU/mL and spore counts were non-detectable [30]. The low microbial load and absence of spores are indicative of excellent hygienic handling and pasteurization effectiveness, which are critical for ensuring consistent fermentation, reproducibility, and reliable assessment of microbial interventions like inulin addition. These values meet the acceptable standards for cheese milk and minimize unwanted microbial interference during ripening [31].

3.1.2. Microbial Quality of Soluble Fiber (Inulin)

The microbial quality of inulin powder was evaluated to ensure it would not introduce microbial contaminants into the cheese matrix. The analysis revealed a very low total plate count (0.99 ± 0.10 Log₁₀ CFU/g), with coliform levels ≤ 10 CFU/g and no detectable spores (

Table 2. Microbial Quality of Soluble Fiber (Inulin) used for incorporation in Cheese.

Parameters
TPC (Log10 CFU/g)	0.99 ± 0.10
Coliforms (Log10 CFU/g)	≤1.0 log
Spores (Log10 CFU/g)	BDL

Below detection limit (BDL)—spore counts < 1 Log₁₀ CFU/mL; TPC—Total Plate Counts.

), indicating that the inulin used was microbiologically safe to prevent contamination. These results are crucial because they confirm that any observed microbial shifts during cheese ripening, particularly the enhancement of non-starter lactic acid bacteria (NSLAB) can be confidently attributed to inulin’s prebiotic effect rather than external contamination. Montanuci et al. (2012) also highlighted that prebiotic integration does not adversely affect microbial safety when properly sourced [32]. Thus, this clean microbial profile supports the suitability of inulin as a clean-label additive for modulating cheese microflora while aligning with food safety standards and consumer demand for natural functional foods.

3.1.3. Total Plate Counts (TPC) of Cheese Samples with Added Inulin

Monitoring total microbial load during cheese ripening offers insight into the overall microbial stability and health of the cheese matrix. At the beginning of ripening (Month zero), TPC ranged from 8.57 ± 0.02 to 8.90 ± 0.04 Log₁₀ CFU/g, indicating robust microbial activity consistent with post-manufacturing conditions. A slight increase was observed during first month up to 9.4 Log₁₀ CFU/g, after which TPC stabilized through third month, and a significant rise was detected from month four onward, where TPC values sharply increased to over 12.4 Log₁₀ CFU/g, and this trend continued consistently till six months. Importantly, no significant differences (p > 0.05) were observed between treatments from fourth to sixth month, suggesting that the presence of soluble fiber maintained microbial stability across all treatments, regardless of spore inoculation (

Table 3. Total plate counts (TPC) (Log₁₀ CFU/g) on Tryptic soy agar (TSA) of different cheese samples stored at 7 °C for ripening.

Duration	Treatments
(Months)	T1 (C SF)	T2 (BL SF)	T3 (CT SF)	T4 (BL+CT SF)
0	8.57 ± 0.02 cC	8.73 ± 0.05 bC	8.79 ± 0.03 bC	8.90 ± 0.04 aC
1	9.42 ± 0.01 aB	9.46 ± 0.03 aB	9.42 ± 0.03 aB	9.43 ± 0.02 aB
2	9.45 ± 0.03 aB	9.51 ± 0.09 aB	9.48 ± 0.07 aB	9.45 ± 0.01 aB
3	9.41 ± 0.01 aB	9.37 ± 0.01 aB	9.37 ± 0.04 aB	9.38 ± 0.04 aB
4	12.41 ± 0.03 aA	12.42 ± 0.03 aA	12.44 ± 0.02 aA	12.41 ± 0.03 aA
5	12.41 ± 0.05 aA	12.44 ± 0.01 aA	12.43 ± 0.01 aA	12.40 ± 0.01 aA
6	12.40 ± 0.02 aA	12.39 ± 0.03 aA	12.38 ± 0.03 aA	12.37 ± 0.01 aA

Different superscripts with small letters (a–c) within a column differ significantly (p < 0.05); Different superscripts with capital letters (A–C) within a column differ significantly (p < 0.05). T1 (C SF)—Control cheese with soluble fiber; T2 (BL SF)—Cheese inoculated with aerobic spores (B. licheniformis) with soluble fiber; T3 (CT SF)—Cheese inoculated with anaerobic spores (Cl. tyrobutyricum) with soluble fiber; T4 (BL+CT SF)—Cheese inoculated with aerobic and anaerobic spores (B. licheniformis & Cl. tyrobutyricum) with soluble fiber.

). Similar results were observed by Kunova et al. (2015) and Carminati et al. (2023), where total counts increased during ripening, indicating microbial proliferation over time [4,33]. This increase is typical during cheese ripening, reflecting the growth of non-starter lactic acid bacteria (NSLAB) and other microorganisms that contribute to the development of cheese flavor and texture. These values were higher than those observed in cheese made without soluble fiber, where TPC plateaued around 9.5 Log₁₀ CFU/g [30], likely due to less support for microbial proliferation in the absence of prebiotic stimulation [30]. The TPC profile indicates that soluble fiber enhanced microbial activity, possibly by stimulating NSLAB growth, which is known to increase in mid-to-late ripening. The rise in microbial load did not coincide with spoilage symptoms, which suggests that the increased activity was largely due to beneficial fermentative bacteria. Moreover, the similarity in TPC across spore-inoculated and control treatments supports the hypothesis that inulin supports overall microbial growth without favoring spoilage organisms, maintaining the microbial balance in the cheese matrix. This aligns with previous findings where prebiotics helped maintain beneficial microbiota in fermented dairy systems [13,34].

3.1.4. Changes in Spore Counts of Cheese Samples with Added Inulin

In cheeses formulated with inulin, spore counts remained consistently low across all treatments throughout six months of ripening. For instance, in the T4 (BL+CT SF), spore counts increased modestly from 0.523 Log₁₀ CFU/g at first month to 0.712 ± 0.025 Log₁₀ CFU/g after six months. Similarly, T1 (CT SF) and T2 (BL SF) samples exhibited final counts of 0.912 ± 0.03 and 0.773 ± 0.013 Log₁₀ CFU/g, respectively, with all spore levels remaining below 1.0 Log₁₀ CFU/g, indicating effective suppression throughout maturation. By contrast, cheese samples manufactured without inulin showed significantly higher spore proliferation where in the cheese samples inoculated with mixture of spores without soluble fiber, spore counts rose from 1.021 ± 0.088 Log₁₀ CFU/g at first month to 2.483 ± 0.028 Log₁₀ CFU/g after six months [30]. The highest spore levels were recorded in the cheese samples inoculated with spores of Cl. tyrobutyricum, which reached 2.941 ± 0.020 Log₁₀ CFU/g, while cheese samples inoculated with spores of B. licheniformis also increased to 2.666 ± 0.008 Log₁₀ CFU/g at the end of ripening (Figure 1). These levels exceeded the spoilage threshold typically associated with late-blowing defects, consistent with visual observations of slits, cracks, and gas formation after five months in the cheese samples without inulin. The comparative data demonstrate that the addition of inulin markedly limited spore growth under ripening conditions. This is likely due to its prebiotic properties, which selectively enhance the growth and metabolic activity of NSLAB, thereby improving microbial competition and limiting nutrient availability to spore-formers. These findings align with previous work by Hegab et al. (2021), who reported improved microbial safety in inulin-enriched Karish cheese [19]. The marked contrast in spore dynamics between treatments further supports the potential of soluble fibers, such as inulin, as a natural intervention strategy for suppressing spoilage organisms in semi-hard cheeses during long-term ripening.

3.1.5. Starter and NSLAB Counts of Cheese Samples with Added Inulin

Starter counts, enumerated on M17 agar, exhibited a characteristic decline during the six-month ripening period across all cheese types (

Table 4. Starter counts (Log₁₀ CFU/g) on M17 agar of different cheese samples stored at 7 °C for ripening.

Duration	Treatments
(Months)	T1 (C SF)	T2 (BL SF)	T3 (CT SF)	T4 (BL+CT SF)
0	8.03 ± 0.12 aA	8.12 ± 0.18 aA	8.01 ± 0.06 aA	8.14 ± 0.23 aA
1	7.36 ± 0.0 aB	6.40 ± 0.02 bB	6.36 ± 0.03 bB	6.37 ± 0.01 bB
2	3.94 ± 0.01 aC	3.74 ± 0.17 aC	3.34 ± 0.05 bC	3.42 ± 0.06 bC
3	2.73 ± 0.13 bD	2.64 ± 0.10 bD	2.62 ± 0.01 bD	3.05 ± 0.42 aD
4	0.92 ± 0.06 aE	0.88 ± 0.12 aE	0.91 ± 0.09 aE	0.90 ± 0.14 aE
5	0.42 ± 0.04 aF	0.40 ± 0.08 aF	0.42 ± 0.17 aF	0.40 ± 0.23 aF
6	0.10 ± 0.43 bF	0.15 ± 0.15 bF	0.20 ± 0.23 aF	0.22 ± 0.19 aF

Different superscripts with small letters (a,b) within a row differ significantly (p < 0.05). Different superscripts with capital letters (A–F) within a column differ significantly (p < 0.05). T1 (C SF)—Control cheese with soluble fiber; T2 (BL SF)—Cheese inoculated with aerobic spores (B. licheniformis) with soluble fiber; T3 (CT SF)—Cheese inoculated with anaerobic spores (Cl. tyrobutyricum) with soluble fiber; T4 (BL+CT SF)—Cheese inoculated with aerobic and anaerobic spores (B. licheniformis & Cl. tyrobutyricum) with soluble fiber.

). Initial SLAB levels were high, ranging from 8.01 ± 0.06 to 8.14 ± 0.23 Log₁₀ CFU/g at month zero, reflecting the expected dominance of the starter culture post-manufacture. After first month, a significant reduction was observed, particularly in spore-inoculated treatments T2, T3, T4, where counts fell to approximately 6.36–6.40 Log₁₀ CFU/g, compared to 7.36 ± 0.0 in the control T1 (C SF). This trend continued with progressive decline through third month, dropping below 3.0 Log₁₀ CFU/g, and nearing extinction (≤0.22 Log₁₀ CFU/g) after six months. The decline was most rapid in the T3 (CT SF) and T2 (BL SF) samples, likely due to environmental stress induced by spore activity and competitive interactions. However, no significant differences were observed in the final counts among treatments, indicating uniform starter exhaustion due to acidification, substrate depletion, and accumulation of inhibitory metabolites, consistent with findings by Fox et al. and Williams et al., 2000, where counts started to decline after one month of ripening and went below 1 Log₁₀ CFU/g after six months of ripening [35,36].

NSLAB counts, assessed on MRS agar, increased steadily during ripening in all samples. Initial populations ranged from 1.99 ± 0.05 to 2.43 ± 0.11 Log₁₀ CFU/g. By the third month, NSLAB counts surpassed 9.3 Log₁₀ CFU/g in all samples, reaching their peak between four to six months. The highest final counts were observed in the control (10.61 ± 0.05 Log₁₀ CFU/g), while T3 (CT SF) samples exhibited slightly lower levels (10.49 ± 0.04), though not significantly different (p > 0.05). Notably, inulin-fortified cheeses maintained significantly higher NSLAB counts compared to the same treatments in the earlier phase without fiber [30]. For example, NSLAB in the control cheese without inulin maintained around 8.5 Log₁₀ CFU/g by third month, whereas the control with inulin (T1) reached over 10.6 Log₁₀ CFU/g after third month (Figure 2). This confirms the enhancement effect of soluble fiber on NSLAB proliferation during ripening. The T2 (BL SF) and T4 (BL+CT SF) samples also recorded robust NSLAB levels (>10.5 Log₁₀ CFU/g), despite the presence of spore-formers, suggesting that inulin supports microbial resilience and competitiveness for NSLAB. These results align with previous findings by Hegab et al. (2021), who observed increased LAB viability and microbial safety in inulin-fortified Karish cheese [19]. Similarly, Gänzle and Follador (2012) reported that inulin-type fructans selectively promoted Lactobacillus species in dairy matrices, enhancing functional properties without compromising quality [37]. Furthermore, Santos et al. (2015) demonstrated that inulin addition led to a higher abundance of Lactobacillus spp. in ripened Minas cheese [38]. Overall, Cheeses fortified with inulin exhibited higher NSLAB levels throughout ripening compared to those without, indicating that inulin enhanced their growth. In Figure 2, the solid lines represent mean NSLAB counts over ripening, while vertical bars indicate standard deviations (±SD) from triplicate analyses. Statistical differences between treatments and ripening stages were evaluated using two-way ANOVA, followed by Tukey’s HSD post hoc test (p < 0.05). Overall, cheeses fortified with inulin exhibited significantly higher NSLAB levels throughout ripening compared to those without inulin, indicating that soluble fiber provides a growth advantage to NSLAB. This suggests that while SLAB behavior remains unaffected, inulin provides a distinct advantage to NSLAB populations, strengthening their persistence and potentially supporting their role in inhibiting spoilage organisms.

3.1.6. Percent Distribution of NSLAB by MALDI-TOF During Ripening

The species-level distribution of non-starter lactic acid bacteria (NSLAB) during six months of cheese ripening revealed marked differences between treatments with and without inulin, particularly in the dominance and stability of key species. Without inulin, Lb. rhamnosus consistently emerged as the most dominant NSLAB across all treatments after six months, accounting for 37% in both Control and cheese with anaerobic spores of Cl. tyrobutyricum, and 35% in samples with aerobic spores and mixture of spores [30]. In contrast, with inulin, the dominance of Lb. rhamnosus was further enhanced, reaching up to 46% by fourth month in the control with soluble fiber (C SF) and stabilizing at 41% after six months. Similar increases were noted in the inulin-fortified T2, T3, and T4 cheeses (Figure 3), where Lb. rhamnosus reached 45%, 40%, and 44%, respectively, clearly exceeding the levels observed without fiber. For Lb. paracasei, a similar enhancement trend was evident. In non-inulin cheeses, their relative abundance ranged from 20% to 26% across treatments, whereas inulin-enriched variants showed a consistent rise, reaching 32% in T1 and 23 to 29% in the remaining fiber treatments by the sixth month. The third and fourth most represented species, Leuconostoc mesenteroides and Lb. plantarum, showed greater stability in the fiber-supplemented cheeses as well, though their proportions were generally lower (5–13%). Notably, a significant difference was observed in the “Other” category: in non-inulin samples, it accounted for a higher and more variable fraction of the community (19–34%), indicating a more heterogeneous and less selective NSLAB population [30]. In contrast, with inulin, this proportion declined and stabilized over time (as low as 11 to 12% after six months), reflecting the selective enrichment of dominant NSLAB species. The species-level distribution results highlight the selective influence of inulin on the NSLAB community during cheese ripening. Inulin significantly enhanced the dominance of beneficial and functionally important NSLAB species, particularly Lactobacillus rhamnosus and Lb. paracasei across all treatments, including those challenged with spore-formers. The increased proportion of these two species, both of which are known for their proteolytic activity, acid tolerance, and potential antimicrobial properties [17,39], suggests that inulin not only promotes their growth but may also help stabilize the microbial ecosystem during long ripening periods. The reduction in the proportion of the “Other” category in inulin-treated cheeses further indicates that inulin selectively enriches specific NSLAB rather than promoting broad microbial growth. This targeted enrichment supports a more controlled and predictable microbial environment, reducing heterogeneity and potentially minimizing the presence of opportunistic or spoilage-associated species.

3.2. Physicochemical Analysis

3.2.1. Moisture Content During Cheese Ripening

Moisture content decreased progressively across all cheese samples over the six-month ripening period, with values ranging from 39.73 ± 1.76% at the first month to 35.88 ± 1.30% after six months (

Table 5. Moisture content (%) of different cheese samples stored at 7 °C for ripening.

Duration	Treatments
(Months)	T1 (C SF)	T2 (BL SF)	T3 (CT SF)	T4 (BL+CT SF)
1	38.08 ± 0.58 cA	38.96 ± 0.10 bA	39.15 ± 0.67 bA	39.73 ± 1.76 aA
2	39.37 ± 0.44 aA	38.52 ± 0.19 bA	37.63 ± 1.73 cB	38.99 ± 0.85 bB
3	38.01 ± 0.44 aB	36.94 ± 0.79 cB	37.83 ± 1.36 bB	37.77 ± 1.55 bC
4	36.31 ± 0.29 bC	36.73 ± 0.80 bB	36.48 ± 1.31 bC	37.04 ± 1.22 aD
5	36.93 ± 0.89 bD	36.38 ± 0.45 bC	37.62 ± 1.02 aB	36.93 ± 0.93 bD
6	36.13 ± 0.99 cE	35.88 ± 1.30 bD	36.63 ± 0.60 aC	36.91 ± 0.09 aD

Different superscripts with small letters (a–c) within a row differ significantly (p < 0.05). Different superscripts with capital letters (A–E) within a column differ significantly (p < 0.05). T1 (C SF)—Control cheese with soluble fiber; T2 (BL SF)—Cheese inoculated with aerobic spores (B. licheniformis) with soluble fiber; T3 (CT SF)—Cheese inoculated with anaerobic spores (Cl. tyrobutyricum) with soluble fiber; T4 (BL+CT SF)—Cheese inoculated with aerobic and anaerobic spores (B. licheniformis & Cl. tyrobutyricum) with soluble fiber.

). The highest initial moisture was observed in the T4 (BL+CT SF) cheese (39.73 ± 1.76%), while the lowest final value was in T2 (BL SF) (35.88 ± 1.30%). The control T1 (C SF) moisture declined from 38.08 ± 0.58% to 36.13 ± 0.99%, showing a typical ripening-related reduction attributed to syneresis and evaporation under controlled storage at 7 °C. The T3 (CT SF) samples exhibited slightly higher moisture retention after six months (36.63 ± 0.60%), likely due to internal gas formation caused by anaerobic spore metabolism, which may interfere with curd shrinkage and drainage [40]. When compared with the cheese manufactured without soluble fiber, the overall moisture decline followed a similar trajectory, although cheeses with inulin tended to retain slightly more moisture in the early stages (one to two months), possibly due to the water-binding properties of inulin [16]. This aligns with previous findings that soluble fibers, such as inulin, can influence water-holding capacity and textural properties in cheese matrices [16,41,42]. However, after six months, the differences were not statistically significant (p > 0.05), and all samples converged within the expected moisture range for semi-hard cheeses (34–37%) [34,43]. The observed moisture decline is consistent with typical cheddar ripening patterns [1]. Inulin did not significantly alter the overall dehydration process, suggesting its inclusion does not compromise moisture regulation. Slightly higher standard deviations in T3 (CT SF) and T4 (BL+CT SF) groups indicate potential localized effects from spore activity, but the general trend was unaffected, ensuring comparability across treatments.

3.2.2. Protein Content of Cheese Samples During Ripening

Protein content remained relatively stable across all cheese samples, ranging from 23.78 ± 0.19% to 25.36 ± 0.54% over the six-month ripening period (

Table 6. Protein content (%) of different cheese samples stored at 7 °C for ripening.

Duration	Cheese Categories
(Months)	T1 (C SF)	T2 (BL SF)	T3 (CT SF)	T4 (BL+CT SF)
1	24.56 ± 0.20	24.07 ± 0.51	24.73 ± 0.49	23.78 ± 0.19
2	24.59 ± 0.13	24.11 ± 0.25	24.52 ± 0.12	23.62 ± 0.28
3	24.38 ± 0.17	24.05 ± 0.16	24.62 ± 0.02	23.63 ± 0.30
4	24.99 ± 0.23	24.81 ± 0.13	24.39 ± 0.26	24.25 ± 0.93
5	24.97 ± 0.65	24.91 ± 0.24	24.77 ± 0.16	24.14 ± 0.16
6	25.20 ± 0.58	25.01 ± 0.22	25.36 ± 0.54	24.65 ± 0.34

Values within rows and columns were not significantly different (p < 0.05). T1 (C SF)—Control cheese with soluble fiber; T2 (BL SF)—Cheese inoculated with aerobic spores (B. licheniformis) with soluble fiber; T3 (CT SF)—Cheese inoculated with anaerobic spores (Cl. tyrobutyricum) with soluble fiber; T4 (BL+CT SF)—Cheese inoculated with aerobic and anaerobic spores (B. licheniformis & Cl. tyrobutyricum) with soluble fiber.

). The T1 (C SF) increased from 24.56 ± 0.20% at the first month to 25.20 ± 0.58% after six months, primarily due to moisture loss rather than proteolysis. Similar trends were observed in T2 (BL SF) and T4 (BL+CT SF), both ending around 25.0 to 25.3%. The T3 (CT SF) group showed slightly higher variation (23.72 to 25.36%), likely reflecting subtle structural disruption or localized proteolytic activity due to anaerobic spore germination. These patterns closely mirror the trends observed in the first phase of the study (without inulin) [30], where protein levels also increased modestly but remained within the expected range for maturing semi-hard cheese. This consistency indicates that neither spore inoculation nor inulin addition significantly altered total protein content, which is expected as proteolysis affects nitrogen solubility and peptide breakdown rather than gross protein percentage [44,45]. The minor increase in protein content is attributed to moisture evaporation rather than active protein synthesis or degradation. The stable protein levels suggest that inulin did not interfere with protein preservation and that any proteolytic activity by NSLAB, or spore-formers was likely masked in overall percentages. These results confirm the structural integrity and consistency of the cheese matrix across treatments during extended ripening.

3.2.3. Fat Content During Cheese Ripening

Fat content remained relatively stable across all cheese samples during the six-month ripening period (

Table 7. Fat content (%) of different cheese samples stored at 7 °C for ripening.

Duration	Treatments
(Months)	T1 (C SF)	T2 (BL SF)	T3 (CT SF)	T4 (BL+CT SF)
1	33.92 ± 0.17	34.18 ± 0.95	34.12 ± 0.92	35.27 ± 0.49
2	34.11 ± 1.03	33.91 ± 0.54	33.28 ± 0.14	34.13 ± 0.24
3	34.53 ± 0.02	34.50 ± 0.33	34.70 ± 0.78	34.02 ± 0.52
4	34.88 ± 0.28	35.03 ± 0.58	33.32 ± 0.36	34.56 ± 0.03
5	33.62 ± 0.93	34.29 ± 0.95	34.55 ± 0.63	34.95 ± 0.70
6	34.19 ± 0.53	33.68 ± 0.19	35.19 ± 0.58	34.92 ± 0.45

All values are presented as mean ± standard deviation; differences were not statistically significant (p > 0.05). T1 (C SF)—Control cheese with soluble fiber; T2 (BL SF)—Cheese inoculated with aerobic spores (B. licheniformis) with soluble fiber; T3 (CT SF)—Cheese inoculated with anaerobic spores (Cl. tyrobutyricum) with soluble fiber; T4 (BL+CT SF)—Cheese inoculated with aerobic and anaerobic spores (B. licheniformis & Cl. tyrobutyricum) with soluble fiber.

), with values ranging from 33.25% to 35.27%. The control T1 (C SF) exhibited minimal fluctuation, starting at 33.92 ± 0.17% in first month and ending at 34.19 ± 0.53% in six months. Similar trends were seen in T2, T3, and T4 cheeses, where no statistically significant changes (p > 0.05) were observed throughout the ripening period.

When compared with the first phase (cheese without soluble fiber), fat contents followed nearly identical trajectories, suggesting that neither inulin incorporation nor spore inoculation impacted the total fat concentration. Minor variations observed across months were attributed to moisture loss and slight redistribution of fat during curd restructuring, rather than lipolytic degradation of triglycerides [1,23]. The consistency in fat content indicates that metabolic activity during ripening, whether from NSLAB or spore-formers, did not significantly affect the gross fat levels. This aligns with the literature stating that microbial lipolysis affects triglyceride breakdown into free fatty acids (FFAs) but not the overall fat percentage [29,46].

3.2.4. Free Fatty Acids and pH During Cheese Ripening

The progression of lipolysis, as measured by free fatty acid (FFA) content (% oleic acid), showed a consistent and gradual increase throughout the six-month ripening period in all cheese samples (

Table 8. Free Fatty Acid (FFA) content (% oleic acid) of different cheese samples stored at 7 °C for ripening.

Duration	Treatments
(Months)	T1 (C SF)	T2 (BL SF)	T3 (CT SF)	T4 (BL+CT SF)
1	0.68 ± 0.04 C	0.63 ± 0.01 C	0.60 ± 0.06 D	0.69 ± 0.01 D
2	0.73 ± 0.03 C	0.73 ± 0.04 C	0.76 ± 0.04 C	0.71 ± 0.05 D
3	0.79 ± 0.02 C	0.80 ± 0.04 B	0.78 ± 0.01 C	0.80 ± 0.00 C
4	0.84 ± 0.06 B	0.82 ± 0.02 B	0.82 ± 0.07 B	0.84 ± 0.01 B
5	0.89 ± 0.01 B	0.87 ± 0.05 B	0.88 ± 0.01 B	0.87 ± 0.02 B
6	0.98 ± 0.06 A	0.99 ± 0.03 A	0.96 ± 0.05 A	0.96 ± 0.02 A

Values within rows were not significantly different (p < 0.05). Different superscripts with capital letters (A–D) within a column differ significantly (p < 0.05). T1 (C SF)—Control cheese with soluble fiber; T2 (BL SF)—Cheese inoculated with aerobic spores (B. licheniformis) with soluble fiber; T3 (CT SF)—Cheese inoculated with anaerobic spores (Cl. tyrobutyricum) with soluble fiber; T4 (BL+CT SF)—Cheese inoculated with aerobic and anaerobic spores (B. licheniformis & Cl. tyrobutyricum) with soluble fiber.

). Initial FFA levels at first month ranged from 0.60 ± 0.06% in T1 (CT SF) to 0.69 ± 0.01% in T4 (BL+CT SF), rising steadily to final values between 0.96 ± 0.02% and 0.99 ± 0.03% after six months. The control T1 (C SF) and T2 (BL SF) treatments exhibited the highest FFA levels (0.98–0.99%), while T3 (CT SF) and T4 (BL+CT SF) remained slightly lower but not significantly different (p > 0.05). Simultaneously, the pH of all cheeses remained remarkably stable across treatments and time points, ranging narrowly between 4.95 and 5.04 throughout the ripening period (

Table 9. pH content of different cheese samples stored at 7 °C for ripening.

Duration	Cheese Categories
(Months)	T1 (C SF)	T2 (BL SF)	T3 (CT SF)	T4 (BL+CT SF)
1	5.00 ± 0.04	4.96 ± 0.00	4.99 ± 0.01	4.98 ± 0.04
2	4.98 ± 0.02	4.97 ± 0.00	4.96 ± 0.02	5.04 ± 0.06
3	5.00 ± 0.01	5.02 ± 0.07	4.98 ± 0.03	4.98 ± 0.07
4	4.99 ± 0.01	5.02 ± 0.02	4.99 ± 0.01	4.99 ± 0.04
5	4.95 ± 0.02	4.98 ± 0.06	5.03 ± 0.03	5.00 ± 0.01
6	5.01 ± 0.03	4.97 ± 0.04	5.03 ± 0.03	4.97 ± 0.01

All values are presented as mean ± standard deviation; differences were not statistically significant (p > 0.05). T1 (C SF)—Control cheese with soluble fiber; T2 (BL SF)—Cheese inoculated with aerobic spores (B. licheniformis) with soluble fiber; T3 (CT SF)—Cheese inoculated with anaerobic spores (Cl. tyrobutyricum) with soluble fiber; T4 (BL+CT SF)—Cheese inoculated with aerobic and anaerobic spores (B. licheniformis & Cl. tyrobutyricum) with soluble fiber.

). This stability was observed in both control and spore-inoculated cheeses, regardless of the presence of inulin. This consistent pH environment provided optimal conditions for NSLAB activity while potentially restricting the outgrowth of pH-sensitive spore-forming spoilage organisms like Cl. tyrobutyricum. The rising FFA levels, alongside pH stability, correspond closely with the sharp increase in NSLAB counts (Figure 2), which peaked between 10.49 and 10.61 Log₁₀ CFU/g after six months. NSLAB species such as Lb. rhamnosus and Lb. paracasei, confirmed by MALDI-TOF profiling, are known producers of esterases and lipases that contribute to secondary lipolysis during cheese maturation [47,48]. The concurrent high FFA levels and robust NSLAB populations suggest a strong metabolic contribution from these organisms, particularly in inulin-enriched samples where microbial competition was better regulated, and spoilage was absent. The stable pH and gradual increase in FFA reflect a well-balanced ripening ecosystem dominated by beneficial NSLAB. The addition of inulin played a dual role in enhancing the growth and activity of NSLAB [49], and indirectly suppressing spore-former activity, resulting in consistent pH control and moderate lipolysis [50]. These findings highlight the importance of microbial dynamics in influencing biochemical ripening markers and confirm that prebiotic fibers, like inulin, can fine-tune ripening processes in favor of quality and safety.

Overall, the results from both microbial and physicochemical analyses demonstrate that the incorporation of inulin into cheese effectively enhanced NSLAB growth and stability while suppressing spore-former proliferation. Cheeses with inulin showed significantly higher NSLAB counts (>10.5 Log₁₀ CFU/g) and maintained low spore loads (<0.9 Log₁₀ CFU/g), with no visible spoilage, unlike non-inulin controls. Stable pH around 5.0 and moderate increases in free fatty acids reflected balanced microbial metabolism and limited lipolytic activity from spore-formers. These findings suggest that inulin not only promotes beneficial microbial succession but also contributes to preserving cheese quality during extended ripening.

4. Conclusions

This study investigated the effect of incorporating inulin, a soluble prebiotic fiber, on the microbial and physicochemical characteristics of Cheddar cheese during six months of ripening, with a particular focus on its potential to enhance non-starter lactic acid bacteria (NSLAB) and suppress the outgrowth of spoilage-associated spore-forming bacteria. The findings provide compelling evidence that inulin can serve as a dual-function additive, supporting both microbial safety and functional quality in ripened cheese. Inulin-containing cheeses demonstrated a clear selective stimulation of NSLAB, especially Lb. rhamnosus and Lb. paracasei, which reached and maintained high viable counts (>10.5 Log₁₀ CFU/g) throughout ripening. This microbial trend was consistent across all treatments, including those inoculated with B. licheniformis (aerobic) and Cl. tyrobutyricum (anaerobic) spores. Importantly, spore counts in inulin-supplemented cheeses remained below 0.9 Log₁₀ CFU/g, and no visible signs of spoilage, such as cracks or late-blowing, were observed, in stark contrast to the first objective (cheeses without inulin), where spore counts reached 3.0 Log₁₀ CFU/g and physical defects became apparent within five months. These results suggest that inulin enhances the ecological competitiveness of NSLAB, enabling them to suppress or outcompete spoilage organisms, likely through acidification, competition for nutrients, and possible bacteriocin production.

Physicochemically, all cheeses with inulin maintained stable pH (~5.0) and protein and fat contents throughout ripening. The progressive but controlled increase in free fatty acids (up to ~0.99% oleic acid) indicated active, but balanced, lipolysis, likely driven by NSLAB enzymes rather than excessive spoilage metabolism. Inulin did not negatively impact cheese composition or ripening behavior, confirming its compatibility with standard cheese manufacturing processes.

Collectively, these findings validate the use of inulin as a clean-label, functional strategy to support beneficial microbial succession and inhibit spoilage in semi-hard cheeses. The ability of inulin to enhance NSLAB activity while suppressing spore-formers opens new avenues for developing safer, shelf-stable, and health-oriented dairy products. Future research should explore strain-specific adjuncts for synergistic effects with soluble fibers and evaluate pilot-scale feasibility for broader commercial application.