Effects of Stinging nettle Powder on Probiotics Survival, Physiochemical Properties, and Nutritional Value of Kefir

Abstract

Kefir is a historic dairy-fermented beverage produced using lactic acid bacteria and yeast as a starter culture and is considered nutritious with a good taste. Many studies have been conducted to incorporate various possible functional materials into kefir to enhance its nutritional value. This study aims to enrich kefir with 0.25% and 0.5% of Stinging nettle (Sn) powder before fermentation to improve its nutritional value. Stinging nettle (Urtica dioica) is a nutritious and multifunctional herb with a variety of healthful components such as fibers and polyphenols; it has significant potential as a useful food functional ingredient. The physicochemical, microbial, and nutritional properties of kefir fortified with Sn were examined weekly during refrigerated storage for 21 days. The results showed that adding Stinging nettle significantly (p < 0.05) increased the probiotic counts from 7.90 ± 0.22 log to 8.46 ± 0.19 log CFU/g, antioxidant activity (4%), and total polyphenol contents (5%) in kefir yogurt after 12 days of refrigerated storage. The addition of Sn also had a positive effect on the acidity of kefir and increased the viscosity and the syneresis to a certain extent. Furthermore, adding Sn increased lactic acid bacteria counts and the production of short-chain fatty acids after in vitro digestion and colonic fermentation. The results of this study indicated the potential use of Sn powder as a functional ingredient in kefir yogurt and other similar products.

1. Introduction

Stinging nettle (Urtica dioica) has been utilized as a wild herb for thousands of years. Stinging nettle (Sn) is a perennial herbaceous plant belonging to the Urticaceae family that has prickly leaves [1]. The potential for Sn medicinal use has been recognized after the fundamental determination of its chemical structure and pharmacological features [2]. Additionally, Sn has been recognized as a food or part of a food that has therapeutic properties and can prevent and treat some diseases [3]. For example, Sn contains biochemically active substances such as phenols, flavonoids, tannins (high-molecular-weight polyphenols), volatile compounds, fatty acids, polysaccharides, isolectins, sterols, terpenes, proteins, vitamins, and minerals, which can help in reducing the free radicals and improving human health [4]. Many studies have attempted to enrich various foods with Sn leaves or powder as a functional ingredient to enhance the nutritional value of foods, such as minerals, dietary fiber, vitamins, and other bioactive compounds [5]. The health benefits of Stinging nettle were mentioned by Kanani [6], who indicated that nettle protected the liver from hepatotoxicity by lowering lipid peroxidation and increasing the antioxidant defense system activity in rats given carbon tetrachloride (CCl₄). The phenolic compounds contained in Stinging nettle are the main reason for this antioxidant property.

Kefir is made by fermenting milk using a culture containing a mixture of “the kefir grains”. This mixed culture contains lactic acid bacteria (lactobacilli, lactococci, leuconostoc), acetic acid bacteria, and many yeast genera that stick to a polysaccharide matrix [7]. The high nutritional value of kefir comes from its rich chemical composition, including more easily digestible proteins, prebiotic oligosaccharides, minerals, vitamins, and other biologically active metabolites such as organic acids and bacteriocins that can produce an effective antimicrobial function to benefit human health [8,9]. Kefir is rich in vitamins B and C, as well as vitamins A and K and carotenoids [10], with thiamine, pyridoxine, and folic acid being particularly abundant [11]. Kefir contains partially digestible proteins that help the body’s digestion and metabolism [12] and high levels of threonine, serine, alanine, lysine, and ammonia compared to milk, as well as other essential amino acids [10]. As for minerals, kefir is a good source of calcium and magnesium, and it is rich in phosphorus, which helps the body utilize carbohydrates, fats, and proteins [13]. Trace elements such as copper, zinc, and iron are also found in kefir [14].

There have been many studies on combining kefir with plant-based ingredients. For example, mint was added to ice cream fermented with kefir culture to investigate its effect on the properties of the ice cream [15]. Another study by Montanuci et al. [16] showed that adding inulin to kefir prepared using fermented skimmed milk increased the pH and syneresis and decreased its titratable acidity during storage. Those studies suggested that the addition of herbal ingredients has the possibility of altering the physicochemical properties, microbial activity, and antioxidant activity of kefir, with the potential to enhance its health benefits. Consequently, this study examined the effects of Stinging nettle powder as a functional ingredient on the physicochemical, microbial, and nutritional value of kefir. This study provided the scientific answer to the question: does enrichment of kefir with Sn powder improve kefir’s nutritional value?

2. Materials and Methods

2.1. Materials

The skimmed milk was purchased from the local supermarket in Melbourne, Australia (ALDI Stores, Melbourne, VIC, Australia). The stinging powder was purchased from Austral Herbs (Kentucky, NSW, Australia). The KEFIR 12 starter culture (eXact^®, a mixed culture of bacteria and yeasts) was provided by Chr. Hansen (Bayswater, VIC, Australia). The media trypticase soy agar (TSA), de Man Rogosa Sharpe (MRS) agar and M17 agar, and bacteriological peptone were purchased from Thermo Fisher Scientific (Thermo Fisher Scientific, 168 Third Avenue, Waltham, MA, 02451, United States. Lines 130–131). Sodium hydroxide (NaOH), Folin–Ciocalteu phenol reagent (FCR), DPPH (2,2-diphenyl-1-picrylhydrazyl), gallic acid, Trolox standard, L-Cysteine HCl, porcine pepsin, pancreatin, mucin, pectin, and bile salts were purchased from Sigma-Aldrich (Castle Hill, NSW, Australia). Dicalcium phosphate (KCl), potassium dihydrogen phosphate (KH₂PO₄), sodium bicarbonate (NaHCO₃), sodium chloride (NaCl), magnesium chloride hexahydrate (MgCl₂(H₂O)₆), and ammonium carbonate ((NH₄)₂CO₃) were purchased from Chem-Supply Pty Ltd. (Huntingdale, VIC, Australia). Hydrochloric acid (HCl), ethanol, methanol, tryptone, soluble starch, yeast extract, pectin, casein, magnesium sulfate heptahydrate (MgSO₄(H₂O)₇), guar gum, calcium chloride (CaCl₂), Tween 80, and all disposable materials were purchased through the University of Melbourne specialist store (Bio21 Institute, Parkville, VIC, Australia). The standard mixtures of SCFAs were purchased from Cayman chemical (Ann Arbor, MI, USA).

2.2. Methods

2.2.1. Preparing Kefir Samples

Kefir samples fortified with Stinging nettle powder were prepared using skimmed milk pasteurized in a hot water bath at 80–85 °C for 30 min [17]. The pasteurized milk was cooled to 42 °C and inoculated with freeze-dried KEFIR 12 probiotic culture (eXact^®) at a concentration of 0.2 g/L and shaken well, then left for 10 min to dissolve completely. Since UV light at 280 nm or less is considered germicidal to most types of microorganisms, Stinging nettle powder was exposed to UV light for about 20 min [18] before it was added at 0%, 0.25%, and 0.5% w/w to the inoculated milk samples and mixed well using the vortex machine (Ultra Turax T25 D S5, IKA, Königswinter, Germany). The applied concentrations of Sn (0.25% and 0.5%) were selected based on some preliminary trails which showed that using > 0.5% affected the kefir color very significantly, and it became very dark green. The inoculated milk was incubated in a thermostatic incubator at 30 °C ± 1 under aerobic conditions for 16 h until the pH reached 4.4–4.6 and then stored at 4 °C for 1, 14, and 21 days [19]. Kefir samples were prepared in two trials using 2 L of milk in each trial, and all measurements were conducted in triplicate.

2.2.2. Preparation of the Agar Medium

The MRS agar was prepared following the supplier’s instructions. A specific weight of MRS agar was added to Milli-Q water (500 mL) and boiled on a hot plate with continuous stirring using a magnetic bar [20]. The boiled MRS agar was autoclaved at 121 °C for 15 min (HANHSIN VD-3041 autoclave, Tullamarine, VIC, Australia) and cooled to 48 °C in a water bath before pouring into Petri dishes. The trypticase soy agar (TSA) and M17 agar were prepared following the same procedures.

2.2.3. Determination of Microbial Survival During Storage Time

The kefir samples were serially diluted (10⁻¹–10⁻⁷) in sterile peptone water, and 0.1 mL of the appropriate dilutions was then spread plated on MRS, M17, and TSA agar. All plates were inoculated in duplicates, and the final counts were reported as a log CFU/g sample. The inoculated plates were incubated at 37 °C for 48 h under anaerobic conditions, using a BB 150 CO₂ incubator (Thermo Fisher Scientific, 168 Third Avenue, Waltham, MA, USA, 02451). The MRS, M17, and TSA media were designated to isolate and enumerate the Lactobacillus sp., Lactococcus sp., and total plate count, respectively [15].

2.2.4. Analysis of Physicochemical Properties

Color and pH Testing

The color of the kefir samples was measured using a portable colorimeter CR-400 (Minolta chromameter CR-400, Osaka, Japan), and the spectral color parameters L* (lightness), a* (green to red), and b* (blue to yellow) of CIELAB color were reported.

The pH determination was performed using a digital pH meter (HI5222, Hanna Instruments Pty Ltd., Melbourne, Australia) following the method of Kaur Sidhu et al. [19].

Titratable Acidity

The titratable acidity of kefir samples was analyzed following the method of Zahid et al. [17]. A mixture of 10 g of yoghurt sample and 90 mL of Mili-Q water was titrated with 0.1 mol/L NaOH solution, and 200 μL of phenolphthalein (1%, w/v) was used as an indicator. The % lactic acid content was calculated using Equation (1):

%TA = F X V₁ M/V₂

(1)

F = Correction factor (=9 for lactic acid);

V₁ = Volume of NaOH;

M = Molarity of NaOH solution;

V₂ = Volume of sample used in the trituration.

Viscosity and Syneresis Determination

Viscosity was measured using the DV1 Digital Viscometer (Brookfield, John Morris Scientific, Deepdene, VIC, Australia) with spindle number 6 at 50 rpm [21]. The spindle was rotated in 40 mL of kefir in a falcon tube, and the values were recorded in centipoise (cP) during the first 5 revolutions of the rotation from triplicate samples. The mean value was determined as the apparent viscosity of the kefir sample.

Determination of whey separation (syneresis) was based on the method of Ranadheera et al. [22] where 20 ± 0.1 g of the kefir sample was carefully transferred into a funnel lined with Whatman Filter Paper No. 4 and left for 2 h. The result was calculated according to Equation (2) and expressed as % syneresis (w/w).

$$\mathrm{Syneresis} \%={\displaystyle \frac{W-Wt-Wr}{W-Wt}}\times 100\%$$

(2)

where W = Total weight of the falcon tube containing the sample, Wt = Weight of the falcon tube after pouring out the sample, Wr = Weight of residue on filter paper.

Determination of Antioxidant Capacity

The kefir sample (40 µL) was mixed with 1960 µL of 70% ethanol, vortexed, and left at room temperature for 15 min for extraction, followed by centrifugation for 10 min (1252.2× g, 4 °C) (Fixed angle rotor FX6100, Allegra X-12R Centrifuge, Beckman Coulter, Inc., Brea, CA, USA). The supernatant was collected for further analysis. Based on the methodology of Zahid et al. [17], the antioxidant activities in all ethanolic extracts were measured using the DPPH. The sample extract (40 µL) was mixed with 260 µL of 0.1 mM DPPH solution in ethanol. The mixture was incubated at room temperature for 30 min before measuring the absorbance at 517 nm using a Multiskan Go 96-well microplate reader (Thermo Fisher Scientific, Waltham, MA, USA). The standard curve (R² = 0.9915) was plotted using Trolox (0 µg/mL–100 µg/mL), and the final results were reported as mg Trolox equivalent (TE)/g sample.

Determination of Total Polyphenol Content

The sample extract prepared for antioxidant analysis was used for the total polyphenol assay. The total polyphenol contents (TPC) in the sample extracts and gallic acid standard solutions were determined by adding 25 μL of each into a 96-well microplate, mixing with 25 μL Folin–Ciocalteu phenol reagent already diluted in water at a 1:3 ratio (v/v), and incubating for 15 min at room temperature [17]. These incubated mixtures were mixed with 200 μL Milli-Q water and 25 μL of 10% (w/v) Na₂CO₃ and incubated for 60 min in the dark to allow color development. The absorbance was measured at 765 nm using a Multiskan Go microplate reader (Thermo Fisher Scientific, Waltham, MA, USA). The total phenolic content was calculated and expressed as mg gallic acid equivalents per gram sample (mg GAE/g) using the gallic acid standard curve.

2.2.5. In Vitro Digestion and Colonic Fermentation

Preparation of In Vitro Digestion Stock Solutions and Basal Media

Stock solutions of simulated gastric fluid (SGF) and simulated intestinal fluid (SIF) were prepared according to the method of Minekus et al. [23]. The stock digestion fluids were prepared using a mixture of the electrolytes (K⁺, Na⁺, Cl⁻, H₂PO₄, HCO₃, Mg²⁺, NH⁺, and Ca²⁺) at different concentrations. The basal medium was prepared as described by Sirisena et al. [24] (2017) where 5 g soluble starch, 5 g peptone, 5 g tryptone, 4.5 g yeast extract, 4.5 g NaCl, 4.5 g KCl, 2 g pectin, 4 g mucin, 3 g casein, 1.5 g NaHCO₃, 0.8 g L-Cysteine HCl, 1.23 g MgSO₄·7H₂O, 1.0 g guar, 0.5 g KH₂PO₄, 0.5 g K₂HPO₄, 0.4 g bile salts, 0.11 g CaCl₂, and 1 mL Tween-80 were dissolved and made up to 1 L in Milli-Q water. The pH was adjusted to 7.0 with 1 M HCl or 1 M NaOH. SGF, SIF stock solutions and basal medium were sterilized using an autoclave at 121 °C for 20 min (HANHSIN VD-3041 autoclave, VIC, Australia). The SGF and SIF stock solutions were stored at −20 °C, and the basal medium was stored in a refrigerator at 4 °C until used.

In Vitro Digestion

Digestion in this experiment consisted of a gastric and small intestinal digestion phases, following the method of Hossain et al. [25] and Minekus et al. [23]. During the gastric digestion phase, 10 mL of the kefir sample was mixed with 7.5 mL of SGF stock solution, 1.6 mL of porcine pepsin (3200–4500 U/mg), and 5 µL of 0.3 M CaCl₂. The pH was adjusted to 3.0 using 1 M HCL and leveled to 20 mL with Milli-Q water, then incubated for 2 h at 37 °C in a shaking incubator (~120 rpm) (ZWYR-240, Labwit, Ashwood, Australia). The gastric digested samples were then mixed with 11 mL of simulated intestinal fluid, 5 mL of porcine pancreatin (800 U/mL), 2.5 mL fresh bile (160 mM), and 40 µL of 0.3 M CaCl₂. The pH was adjusted to 7.0 using NaOH (1 M) and leveled to 40 mL with Milli-Q water. The samples were flushed with N₂ for 30 s before being digested in a shaking incubator (~120 rpm) at 37 °C for 2 h. The digested samples were centrifuged at 6000 rpm, at 4 °C, for 10 min, and the residues were collected to continue the colonic fermentation.

Fecal Slurry Preparation

Ethical approval (ID: 2024-29822-54940-5) was obtained from the Human Ethics Advisory Group at the University of Melbourne before commencing the colonic fermentation. The preparation of human fecal slurry was conducted based on the method of Tzounis et al. [26]. The fresh fecal sample was collected from a 29-year-old healthy adult male donor without any history of antibiotics and probiotics intake in the past three months. About 10 g of that human feces was mixed with 90 g of sterilized pre-N₂ flushed phosphate buffer (pH = 7) in a stomacher bag and homogenized using a stomacher mixer (400 Circulator, Seward, AK, USA) for 2 min. Fecal slurry (10% w/w) was obtained by aseptically filtering the stomached liquid into a pre-N₂ flushed tube and refrigerated until used.

Colonic Fermentation

During the colonic fermentation phase, 5 mL of basal medium and 5 mL of fecal slurry were added to the residue (~5 g) obtained initially from the already performed intestinal digestion. The mixture was thoroughly mixed and then subjected to 1 min nitrogen flush to create anaerobic conditions. The samples were then placed in a shaking incubator at 37 °C, 120 rpm, and triplicate samples were collected at 0, 48, and 72 h for microbial activity testing and short-chain fatty acid (SCFA) analysis. The same procedures were repeated on days 1 and 21 of storage, and the blank sample was prepared using 5 mL fecal slurry and 5 mL basal medium only [25,27]. Microbial viable counts were assessed following the same method mentioned in Section 2.2.3 using MRS, M17, and TSA media.

2.2.6. Analysis of Short-Chain Fatty Acids

Sample Preparation

Kefir samples subjected to in vitro digestion and colonic fermentation were prepared for short-chain fatty acid analysis following the methods of Gu et al. [28] and Loo et al. [29]. Fermented samples (1.5 mL) were centrifuged at 5000 rpm for 15 min at 4°, and 1.0 mL of the supernatant was transferred into a new 10 mL plastic tube and mixed well with 3.5 mL of diluted acid (1% formic acid and 1% orthophosphoric acid), and 4-methylpentanoic acid (internal standard) at a final concentration of 1.59 mmol/L. A small volume (1.5 mL) of that final mixture was then transferred into a GC vial for GC-FID analysis.

Analyses of SCFAs Using GC-FID

The analysis of short-chain fatty acids was conducted using GC-FID, according to the analytical methods of Gu et al. [28]. The gas chromatography (GC) machine (7890B Agilent, Santa Clara, CA, USA) was coupled with a flame ionization detector (FID), an autosampler (7693 Agilent, CA, USA), and an autoinjector (G4513A Agilent, CA, USA). A SGE BP21 capillary column (12 × 0.53 mm internal diameter (ID) with 0.5 µm film thickness, SGE International, Ringwood, VIC, Australia, P/N 054473) and a retention gap kit (including a 2 × 0.53 mm ID guard column, P/N SGE RGK2) were attached. The carrier gas was helium with a flow rate of 14.4 mL/min. The detailed conditions used for GC-FID were as follows: 1 µL sample was injected; the oven temperature was set at 100 °C for 30 s, then increased to 180 °C at 6 °C/min for 1 min, and then increased to 200 °C at 20 °C/min for 10 min; the FID temperature was set at 240 °C; the inlet temperature was set at 200 °C; and the supplemental gases were nitrogen, hydrogen, and air at flow rates of 20, 30, and 300 mL/min, respectively. Short-chain fatty acids were calculated by substituting the sample peak area directly into the following standardized equation [17]:

$$\mathrm{SCFA} \mathrm{concentration} (\mathrm{mmol}/\mathrm{L}) =\left[{\displaystyle \frac{\mathrm{Sample} \mathrm{peak} \mathrm{area} \times \mathrm{dilution} \mathrm{factor}}{\mathrm{SCFA} \mathrm{molecular} \mathrm{weight} \left(\mathrm{g}\right)}}\right]$$

2.2.7. Statistical Analysis

The generated data were analyzed for significant differences between the treatments and sample mean ratings using a one-way analysis of variance (ANOVA) and Fisher’s least significant difference (LSD) (α = 0.05). All statistical analyses were conducted using the XLSTAT^® (Addinsoft, New York, NY, USA) analysis add-in installed on Excel (Microsoft Corporation, Inc., Redmond, WA, USA).

3. Results

3.1. Microbiological Properties of Kefir Samples During Storage

For microbial enumeration tests during storage, MRS agar was used as an indicator medium for Lactobacillus sp., M17 agar for Lactococcus sp., and TSA agar for total plate counts as a control. The results in

Table 1. Changes in the microbial counts (log CFU/g) using MRS, M17, TSA agar in kefir enriched with various concentrations of Stinging nettle powder during refrigerated storage.

Type of Medium	Kefir Sample	Storage Time (Days)
		1	14	21
MRS	Control *	8.59 Aa ± 0.14	8.15 Bb ± 0.06	7.90 Bc ± 0.22
	Kefir + 0.25% Sn	8.61 Aa ± 0.19	8.37 Aab ± 0.11	8.16 Bb ± 0.14
	Kefir + 0.5% Sn	8.76 Aa ± 0.28	8.57 Aa ± 0.21	8.46 Aa ± 0.19
M17	Control *	8.53 Aa ± 0.30	8.25 Bab ± 0.12	8.13 Bb ± 0.17
	Kefir + 0.25% Sn	8.12 Aa ± 0.40	8.43 Aa ± 0.09	8.48 Aa ± 0.18
	Kefir + 0.5% Sn	8.19 Ab ± 0.22	8.52 Aa ± 0.06	8.55 Aa ± 0.26
TSA	Control *	8.91 Ab ± 0.24	9.24 Aa ± 0.08	8.62 Ac ± 0.11
	Kefir + 0.25% Sn	9.16 Aa ± 0.10	8.74 Ba ± 0.04	8.89 Aa ± 0.48
	Kefir + 0.5% Sn	9.19 Aa ± 0.29	8.97 ABab ± 0.28	8.67 Ab ± 0.09

Values are expressed as the mean ± standard deviation (n = 4). Means followed by different capital letters within each day of testing indicate significant differences (p < 0.05). Means followed by different small letters within a row indicate significant differences (p < 0.05) over storage time. * kefir without added Stinging nettle powder.

show that the counts of Lactobacillus sp., Lactococcus sp., and total plate in the control treatments decreased by 0.69, 0.4, and 0.29 log CFU/g, respectively, after 21 days of refrigerated storage. The same data also indicated that adding Sn powder at 0.25% and 0.5% significantly (p < 0.05) enhanced the survival of LAB in kefir within each day of storage (day 1, 14, and 21). However, no significant (p >0.05) differences were detected in the samples treated with 0.25% and 0.5% Sn powder.

Furthermore, the effects of added Sn on Lactococcus sp. and Lactobacillus sp. were similar. The addition of Sn contributed to the protection of these LAB species with significant increases in their counts (p < 0.05) after 14 days of refrigerated storage (Table 1). In contrast, the counts of Lactobacillus sp. and Lactococcus sp. continued to decrease over the 21 days of storage. The final count in the control was 7.90 ± 0.22 log CFU/g in comparison with 8.46 ± 0.19 and 8.55 ± 0.26 in kefir samples enriched with 0.5% Sn after 21 days of refrigerated storage.

The results of the total plate count in the control (8.91 ± 0.24 log CFU/g) were very close to those obtained from individual counts on MRS and M17, (9.16 ± 0.10 and 9.19 ± 0.29, log CFU/g, respectively) on day 1 of refrigerated storage. Similar observations were also recorded on day 21 of storage, with insignificant (p > 0.05) differences between all treatments (Table 1). However, the TPC on day 14 was significantly (p < 0.05) larger in the control (9.24 ± 0.08 log CFU/g) than in kefir enriched with 0.25% (8.74 ± 0.04 log CFU/g) and 0.5% Sn (8.97 ± 0.28 log CFU/g). Such an increment of day 14 could be attributed to the larger initial count used on day 1, which was 8.91 ± 0.24 log CFU/g of the experimental results, and their interpretation, as well as the experimental conclusions that can be drawn.

3.2. Physicochemical Properties of Kefir

3.2.1. pH Value and Titratable Acidity

pH and titratable acidity are important parameters in determining the quality of kefir yogurt products. From the results in

Table 2. Changes in the physicochemical properties in kefir enriched with various concentrations of Stinging nettle powder during refrigerated storage.

(a)
Testing Indicators	Kefir Sample			Storage Time (Days)
				1		14		21
pH	Control *			4.57 Aa ± 0.01		4.50 Ab ± 0.01		4.32 Ac ± 0.01
	Kefir + 0.25% Sn			4.57 Aa ± 0.01		4.45 Bb ± 0.02		4.28 Bc ± 0.02
	Kefir + 0.5% Sn			4.55 Ba ± 0.01		4.42 Cb ± 0.01		4.30 ABc ± 0.01
Titratable acidity (%TA)	Control *			0.73% Bb ± 0.03		0.77% Bab ± 0.05		0.83% Ca ± 0.02
	Kefir + 0.25% Sn			0.82% Ab ± 0.01		0.90% Aa ± 0.02		0.91% Ba ± 0.02
	Kefir + 0.5% Sn			0.85% Ab ± 0.01		0.95% Aa ± 0.01		0.96% Aa ± 0.02
Viscosity cP (mPs/s)	Control *			3833 Ca ± 577		4433 Ba ± 208		4533 Ba ± 153
	Kefir + 0.25% Sn			4933 Bb ± 462		5700 Bab ± 854		6533 Aa ± 808
	Kefir + 0.5% Sn			6567 Aa ± 493		8866 Aa ± 1629		7833 Aa ± 1185
Syneresis	Control *			67.26% Ab ± 0.01		68.04% Aab ± 0.01		69.02% Aa ± 0.01
	Kefir + 0.25% Sn			67.89% Aa ± 0.01		67.90% Aa ± 0.01		68.25% Aa ± 0.02
	Kefir + 0.5% Sn			68.30% Aa ± 0.01		67.91% Aa ± 0.01		69.79% Aa ± 0.01
(b)
Kefir Sample	Storage Time (Days)
	1			14			21
	L*	-a*	b*	L*	-a*	b*	L*	-a*	b*
Control *	54.94 Ab ± 0.12	1.98 Aa ± 0.04	3.35 Cb ± 0.04	56.92 Aa ± 0.12	1.97 Aa ± 0.01	3.67 Ca ± 0.01	55.11 Ab ± 0.06	1.96 Aa ± 0.01	3.29 Cc ± 0.02
Kefir + 0.25% Sn	50.08 Ba ± 0.46	1.70 Ba ± 0.02	5.16 Bb ± 0.07	47.87 Bb ± 0.08	1.58 Bb ± 0.03	5.26 Ba ± 0.05	47.61 Bb ± 0.03	1.25 Cc ± 0.01	5.00 Bc ± 0.01
Kefir + 0.5% Sn	46.08 Ca ± 0.13	1.51 Ca ± 0.01	5.92 Aa ± 0.07	43.44 Cc ± 0.08	1.42 Cb ± 0.04	5.86 Aa ± 0.01	43.89 Cb ± 0.16	1.33 Bc ± 0.01	5.20 Ab ± 0.09

(a) pH, titratable acidity, viscosity, and syneresis; (b) values are expressed as the mean ± standard deviation (n = 3). Means followed by different capital letters within each day of testing indicate significant differences (p < 0.05). Means followed by different small letters within a row indicate significant differences (p < 0.05) over storage time. * kefir without added Stinging nettle powder. Changes in color (L*—lightness, a*—red/green, b*—blue/yellow) in kefir enriched with various concentrations of Stinging nettle powder during refrigerated storage.

, the pH values of all the products were within the ideal range for kefir products after 16 h of fermentation (4.4–4.6), with a value of 4.57 $\pm$0.01 for the control and kefir enriched with 0.25% Sn, while kefir fortified with 0.5% Sn had a pH 4.55 $\pm$0.01 on day of storage. All kefir samples showed a significant decrease (p < 0.05) in pH during storage, which ranged from 4.55 to 4.30 (Table 2). The pH values in kefir samples with 0.5% Sn were significantly the lowest (p < 0.05) on days 1 and 14, while kefir with the 0.25% Sn sample had the lowest pH value on day 21.

Titratable acidity conformed to the expected negative correlation with pH and gradually increased with the TA% of the samples during storage (range from 0.73–0.85% to 0.83–0.96%), showing a significant increase (p < 0.05) on day 14, and the addition of Sn powder significantly increased the accumulation of titratable acid in kefir yogurt.

3.2.2. Viscosity and Syneresis

Viscosity and syneresis are also important quality indicators of yoghurt. From the results in Table 2, it was found that the viscosity of all the kefir samples increased with storage time. The kefir samples with added Sn showed significantly (p < 0.05) greater viscosity (4933 $\pm$462 and 6567 $\pm$493, for kefir with 0.25 and 0.5%, respectively) than the control (3833 $\pm$577) on day 1, and the same trend was recorded throughout the storage period.

As for the syneresis indicator, its values did not show statistically significant differences (p > 0.05) among all the treatments (Table 2). The % syneresis ranged from the lowest value (67.26 $\pm$0.01) in the control of the day to the largest (69.79% $\pm$0.01 kefir containing 0.5% Sn on day 21.

3.2.3. Color

The color of fermented milk is one of the main quality characteristics that influence consumer acceptance and product attractiveness [30]. The color variations of the samples in this study were analyzed by measuring the L*, a*, and b* values, where the L* value represents brightness, the -a* value represents green, and the b* value represents yellow. The results in

Table 3. Changes in the antioxidant capacity (mg TE/g) in kefir enriched with various concentrations of Stinging nettle powder during refrigerated storage.

Kefir Sample	Storage Time (Days)
	1	14	21
Control *	1.27 Ba ± 0.06	0.82 Bb ± 0.03	0.73 Bc ± 0.02
Kefir + 0.25% Sn	1.31 Ba ± 0.09	0.87 ABb ± 0.06	0.85 Ab ± 0.04
Kefir + 0.5% Sn	1.45 Aa ± 0.08	0.93 Ab ± 0.03	0.77 ABc ± 0.09

Values are expressed as the mean ± standard deviation (n = 4). Means followed by different capital letters within each day of testing indicate significant differences (p < 0.05). Means followed by different small letters within a row indicate significant differences (p < 0.05) over storage time. * kefir without added Stinging nettle powder.

show that the L* values of all the kefir yogurts decreased during storage; the addition of Sn significantly decreased (p < 0.05) the brightness of kefir, and the larger the addition of Sn the more significant the decrease (p < 0.05) in brightness. For the a* values, the a* of the control group did not change significantly (p > 0.05) during storage, whereas the samples with the addition of Sn turned gradually redder during storage, with significant differences (p < 0.05) between days. The addition of Sn significantly reduced the green color in kefir yogurt, bringing the -a* values closer to 0, with higher additions having more significant effects (p < 0.05) on days 1 and 14. For the b* values, it appeared that samples possessed more yellow color (larger b value) after 14 days of storage, with appositive correlation between added amounts of Sn and increases in b values (Table 3).

3.2.4. Changes in Antioxidant Activity and Total Polyphenol Content

The results for the changes in antioxidant activity and total polyphenol content in kefir yogurt enriched with Sn are shown in Table 3 and

Table 4. Changes in the total polyphenolic content (TPC) (mg GAE/g) in kefir enriched with various concentrations of Stinging nettle powder during refrigerated storage.

Kefir Sample	Storage Time (Days)
	1	14	21
Control *	0.21 Ba ± 0.02	0.20 Aa ± 0.03	0.17 Aa ± 0.05
Kefir + 0.25% Sn	0.24 ABa ± 0.02	0.25 Aa ± 0.02	0.22 Aa ± 0.01
Kefir + 0.5% Sn	0.25 Aa ± 0.02	0.23 Aa ± 0.09	0.19 Aa ± 0.03

Values are expressed as the mean ± standard deviation (n = 4). Means followed by different capital letters within each day of testing indicate significant differences (p < 0.05). Means followed by different small letters within a row indicate significant differences (p < 0.05) over storage time. * kefir without added Stinging nettle powder.

, respectively. The results in Table 3 reveal that the antioxidant activity of all the samples decreased during storage. The antioxidant activity on day one decreased significantly (p < 0.05) from 1.27 $\pm$0.06 mg TE/g to 0.73 $\pm$0.02 mg TE/g on day 21 in the control samples, while it decreased from 1.31 $\pm$0.09 mg TE/g to 0.85 $\pm$0.04 mg TE/g in the kefir enriched with 0.25% Sn during the same storage period. A similar trend was also detected in kefir samples with 0.5% Sn. However, the addition of Sn at both 0.25% and 0.5% improved the antioxidant activity significantly (p < 0.05) in kefir compared with the controls.

Unlike the results of the antioxidants activities, the total polyphenol content (TPC) in all treatments did not show significant changes (p > 0.05) during storage (Table 4). However, the TPCs in kefir enriched with Sn were significantly (p < 0.05) larger than those in the control samples. The TPC in the control samples ranged from 0.21 $\pm$0.02 on day 1 of storage to 0.17 $\pm$0.05 mg GAE/g kefir on day 21, while the TPC in kefir enriched with 0.25% Sn ranged from the highest 0.25 $\pm$0.02 on day 14 to the lowest 0.22 $\pm$0.01 mg GAE/g on day 21. Similar TPCs were also reported in kefir enriched with 0.5% Sn. The data in Table 4 and

Table 5. Changes in the microbial count (log CFU/g, on MRS, M17, TSA agar) during colonic fermentation of kefir samples enriched with various concentrations of Stinging nettle powder and stored refrigerated for 1 day.

Type of Medium	Kefir Sample	Fermentation Time (Hours)
		0 h	48 h	72 h
MRS	Control *	3.25 Bc ± 0.22	8.49 Bb ± 0.03	8.9 Ba ± 0.18
	Kefir + 0.25% Sn	3.85 Ab ± 0.33	8.95 Aa ± 0.18	9.20 Aa ± 0.14
	Kefir + 0.5% Sn	3.95 Ab ± 0.26	9.07 Aa ± 0.09	9.37 Aa ± 0.20
M17	Control *	4.18 Bc ± 0.37	8.75 Cb ± 0.10	9.91 Aa ± 0.13
	Kefir + 0.25% Sn	5.01 Ab ± 0.07	9.07 Ba ± 0.15	9.12 Ba ± 0.11
	Kefir + 0.5% Sn	5.19 Ab ± 0.08	8.83 BCa ± 0.09	8.95 Ba ± 0.01
TSA	Control *	5.26 Bb ± 0.49	9.14 Ba ± 0.14	9.55 Ca ± 0.12
	Kefir + 0.25% Sn	5.42 ABc ± 0.33	9.40 ABb ± 0.33	10.50 Aa ± 0.07
	Kefir + 0.5% Sn	5.44 ABc ± 0.05	9.40 ABb ± 0.15	10.37 Aa ± 0.26

Values are expressed as the mean ± standard deviation (n = 3). Means followed by different capital letters within each time point of testing indicate significant differences (p < 0.05). Means followed by different small letters within a row indicate significant differences (p < 0.05) during fermentation.

shows that changes in antioxidant activities and TPCs were consistent with continuous decline during storage. Furthermore, the addition of Sn enhanced both the antioxidant activities and TPCs in kefir compared with the control.

3.3. Microbial Survival After In Vitro Digestion and Colonic Fermentation of Kefir

The in vitro studies of kefir samples were conducted on day 1 and day 21 of refrigerated storage in order to detect changes over time.

3.3.1. In Vitro Fermentation After One Day of Refrigerated Storage

The in vitro gastric digestion phase was conducted at pH 3 for 2 h, followed by intestinal digestion at pH 7 for an additional 2 h. The digested samples were then centrifuged at 6000 rpm at 4 °C for 10 min, and the residues were collected to continue the colonic fermentation. The microbial survival was assessed using three different media: (1) MRS medium to enumerate Lactobacillus sp., (2) M17 to count the Lactococcus sp., and (3) TSA for total plate count [15]. The microbial survival revealed a significant (p < 0.05) decline in the viable counts after gastric and intestinal digestion. The average counts of lactic acid bacteria (LAB) and total plate counts decreased by 4–5 log CFU/g during these phases of digestion. However, the results in Table 5 revealed that within the first 48 h of colonic fermentation the microbial counts recovered quickly and increased significantly (p < 0.05) to reach 8.49 ± 0.03, 8.95 ± 0.18, and 9.07 ± 0.09 log CFU/g in the control, the kefir containing 0.25% Sn, and the kefir with 0.5% Sn, respectively, when using MRS agar. Similar results were also detected using the M17 agar medium that was designated to isolate Lactococcus sp.

The Lactobacillus sp. counts (log CFU/g) in all the samples were the highest after 48 hs and 72 h of fermentation, with insignificant (p > 0.05) differences in the counts after 48 h and 72 h of fermentation. The addition of Sn during fermentation significantly increased (p < 0.05) the viability of Lactobacillus sp. However, adding Sn into kefir did affect the Lactococcus sp. counts during colonic fermentation. Furthermore, the results showed no significant (p > 0.05) differences in microbial counts between kefir samples enriched with 0.25% and 0.5% Sn powder (Table 5).

The total plate counts for all the samples continued to rise throughout the fermentation process, with higher total plate counts in samples with Sn added in comparison with control samples. The total plate counts exceeded 10 log CFU/g after 72 h of colonic fermentation in samples enriched with 0.25% and 0.5% Sn.

3.3.2. In Vitro Fermentation After 21 Days of Refrigerated Storage

The in vitro colonic fermentation and the surviving Lactobacillus sp., Lactococcus sp., and total plate counts in the kefir samples in refrigerated storage for 21 days are shown in

Table 6. Changes in the microbial count (log CFU/g, on MRS, M17, TSA agar) during colonic fermentation of kefir samples enriched with various concentrations of Stinging nettle powder in refrigerated storage for 21 days.

Type of Medium	Kefir Sample	Fermentation Time (Hours)
		0 h	48 h	72 h
MRS	Control *	3.99 Ab ± 0.03	8.97 Aa ± 0.43	9.00 Aa ± 0.18
	Kefir + 0.25% Sn	4.08 Ab ± 0.37	9.00 Aa ± 0.47	9.13 Aa ± 0.27
	Kefir + 0.5% Sn	4.10 Ab ± 0.43	9.02 Aa ± 0.16	9.10 Aa ± 0.20
M17	Control *	4.22 Bb ± 0.49	9.50 Aa ± 0.24	9.39 Aa ± 0.29
	Kefir + 0.25% Sn	5.02 Ab ± 0.10	9.46 ABa ± 0.22	9.40 Aa ± 0.18
	Kefir + 0.5% Sn	5.07 Ab ± 0.14	9.16 Ba ± 0.16	9.00 Ba ± 0.12
TSA	Control *	6.26 Ab ± 0.20	9.62 Aa ± 0.26	9.45 Aa ± 0.38
	Kefir + 0.25% Sn	5.01 Bb ± 0.21	9.61 Aa ± 0.27	9.54 Aa ± 0.21
	Kefir + 0.5% Sn	4.94 Bb ± 0.15	9.36 Aa ± 0.13	9.32 Aa ± 0.14

Values are expressed as the mean ± standard deviation (n = 3). Means followed by different capital letters within each time point of testing indicate significant differences (p < 0.05). Means followed by different small letters within a row indicate significant differences (p < 0.05) during fermentation.

. The results revealed that the in vitro fermentation of kefir samples in refrigerated storage for 21 days was similar to that reported before with samples refrigerated for 1 day only. The low counts in kefir samples after gastric digestion were 3.99 ± 0.03, 4.10 ± 0.43, and 4.10 ± 0.43 in the control, Lactobacillus sp., and Lactococcus sp. enriched with 0.25% Sn, respectively. However, these counts increased significantly (p < 0.05) after 48 hs of fermentation and reached about 9 log CFU/g in all treatments. These same increments were also detected after 72 h of fermentation.

3.3.3. Short-Chain Fatty Acid (SCFA) Production After In Vitro Colonic Fermentation

The kefir samples were subjected to in vitro colonic fermentation followed by GC-FID analysis to identify and quantitate the various short-chain fatty acids (SCFAs) released via fermentation. The order of SCFAs from the fatty acids standard mixture and kefir samples is shown in Figure 1A and 1B, respectively. The identified SCFAs in the standard mixture included acetic acid (with the shortest retention time), followed by propionic acid, iso-butyric acid, butyric acid, iso-valeric acid, and valeric acid (with the longest retention time) (Figure 1A). The SCFAs in the kefir sample were considered the same SCFAs as the external standard within 0.1 min of the retention time. Figure 1B shows that the main short-chain fatty acids that appeared consistently in the kefir samples throughout the colonic fermentation were acetic, propionic, iso-butyric, and butyric acids. The SCFA concentrations (mmol/L) were calculated using GC peak area based on the explanation given before in the analyses of SCFAs using GC-FID.

Acetic acid (AA) was the most abundant SCFA in the kefir samples refrigerated for 1 day before colonic fermentation and reached 5 mmol/L after 48 h of fermentation with insignificant (p > 0.05) changes after 72 h of fermentation (Figure 2A). However, the same results showed that adding Sn at 0.25 and 0.5% promoted the production of AA significantly (p < 0.05) in comparison with the control and the blank.

The production of propionic acid (PA) was smaller than acetic acid among all treatments (Figure 2B). The largest amount of PA was 0.02–0.03 mmol/L after 48 h of fermentation. Similar to the observations recorded with AA, adding Sn at both 0.25% and 0.5% improved the production of PA significantly (p < 0.05).

The amounts of iso-butyric acid accumulated in all samples after colonic fermentation ranged from 0.07 mmol/L before fermentation (0 h) to a maximum value of 1 mmole after 48 and 72 h of fermentation. Furthermore, like AA and PA, adding Sn to kefir samples improved the production of iso-butyric acid significantly (p < 0.05). The same date also showed that enriching kefir with 0.5% Sn showed more iso-butyric acid production than 0.25% after 48 and 72 h of fermentation (Figure 2C).

Butyric acid (BA) was the fourth SCFA detected in refermented kefir samples with small quantities similar to those detected with PA (Figure 2B). Unlike the previously reported SCFA, the data in Figure 2D revealed continuous increases in the amounts of BA after 48 h and 72 h of fermentation. The largest amount of detected BA (0.046 mmol/L) was in kefir enriched with 0.5% Sn and after 72 h of fermentation (Figure 2D). Apparently, both PA and BA were released in much smaller quantities than AA and iso-butyric acid.

The released amounts of SCFSs in kefir samples stored for 21 days before fermentation are presented in Figure 3A–D). The trends of production and accumulation of SCFAs were similar to those observations recorded with samples stored for 1 day only. Furthermore, the effect of adding Sn at 0.2% and 0.5% on the production of SCFAs during colonic fermentation of kefir refrigerated for 21 days before in vitro fermentation was also the same. It should also be mentioned that the blank samples, which were prepared by mixing 5 mL fecal slurry and 5 mL basal medium only produced the smallest amounts of all SCFAs after in vitro fermentation of kefir samples stored for 1 day and 21 days. This is expected since the SCAF can be produced by the gut microbiota through fermentation of compounds that are not hydrolyzed during gastric and intestinal digestions.

4. Discussion

4.1. Effect of Sn on the Microbiological Properties of Kefir Samples During Storage

From the results in Table 1, it can be concluded that the lactic acid bacteria (LAB) counts in all treatments were basically greater than 8 log CFU/g, which exceeds the minimum value of probiotics needed to produce therapeutic activity (6 log CFU/g) [31]. These observations indicated that the products were well fermented and met the minimum standards for probiotic labelling set by the scientific profession [32]. The changes in Lactobacillus sp. and Lactococcus sp. during storage in samples supplemented with Sn were close to the results of Öztürk-Yalçın et al. [15] (8 log CFU/g), who used mint as a prebiotic to promote the growth of LAB. The general decline in LAB counts and total plate counts with extended storage may be due to the conversion of lactose to lactic acid in the milk due to continuous fermentation, and the accumulation of excess lactic acid makes the kefir too acidic and overwhelms the growth of bacteria [33].

The promoting effect of Stinging nettle on probiotic activity may be related to its rich polyphenols, dietary fiber, and protein contents. The gut microbiota can ferment the phenolic compounds found in Sn such as chlorogenic acid (3-CQA) and rutin [34]. This fermentation potentially contributes to health-benefiting properties such as homeostasis regulation and gut microbiota alteration [35]. The prebiotic function of Sn was reported by Kheoane et al. [36], who found that Sn could significantly stimulate the growth of L. rhamnosus compared to inulin. That finding was supported by an in vitro study [37], which showed that yoghurt enriched with Sn successfully promoted the viability of another probiotic (Bifidobacterium). Similar observations related to the effects of polyphenols on probiotics were reported by Zahid et al. [17], who mentioned that enriching yoghurt with mango peel powder improved the survival of LAB during storage. Those authors attributed the positive effects on probiotic survival to the presence of phenolic compounds in mango peel powder. Other studies by Patel [38] and Coman et al. [39] attributed the positive effect of Sn on probiotic survival to the combined effect of the phenolic compounds, dietary fiber, and proteins contained in Sn.

4.2. Effect of Added Sn on Physicochemical Properties of Kefir

This study was based on physiochemical and microbial analyses only. No sensory tasting was included due to time limitations and the cost involved, since this project was not funded. The main reason for the development of acidity in kefir is the fermentation of milk by lactic acid bacteria, including Lactobacillus sp. and Lactococcus sp. [40]. Fermentation allows the lactose of the milk to be converted into lactic acid by the probiotics during the fermentation process, leading to an increase in the acidity of the kefir [41]. The effect of Sn on increasing the acidity of kefir was in line with Kaur Sidhu et al. [19], who investigated the effect of chickpea flour on the increase in acidity in the yogurt. The effects of Sn on yoghurt acidity may be derived from the possible function of Sn powder as a prebiotic and supporting the growth of probiotics during the fermentation process, where more probiotics produce richer lactic acid.

The trend of elevated viscosity due to the addition of Sn in this study was in agreement with results reported by Yangilar & Gülhan [42]. However, the viscosity values in this study were relatively higher, which may be due to differences in preparation and testing procedures. The effect of Sn on the viscosity of kefir may be related to its abundance of protein and fiber affecting the water retention capacity of protein and fat globules in kefir [43].

The increase in syneresis in the presence of Sn might be caused by certain ingredients affecting the structure and development of casein and resulting in a weak gel that is unable to retain water. Similar observations were reported in kefir with added amounts of inulin [16]. However, inconsistent with the effect of fiber-rich ingredients such as chickpea flour, dates, and other products on yogurt syneresis reduction as demonstrated in some other studies [19,30], adding Sn did not demonstrate a syneresis reduction effect, which might be attributed to smaller fiber content in Sn.

The trend of the addition of Stinging nettles for the change in color, in terms of L* and b* values, is consistent with the study of Yangilar & Gülhan [42] and may be derived from the yellowish-green color of the Sn leaves. Moreover, a variety of phenolic compounds contained in Sn may interact with known anthocyanins, leading to fluctuations and variations in color, and may also be responsible for this result [44]. The brightness is provided by the scattered white light formed by the fat globules and casein micelles in the yogurt, and the presence of Sn may alter yogurt brightness by affecting their content and structure [39]. However, the changes in a* values contradicted the results of Yangilar & Gülhan [42], who reported increases in -a* values which did not correspond with the green color of the Sn itself. The detected small -a* values in kefir with added Sn in this study might be caused by the precipitation of the added Sn at the bottom of the yoghurt containers.

4.3. Effect of Added Sn on Antioxidant Activity and Total Polyphenol Content in Kefir

Changes in the TPC contents and antioxidant activity in kefir enriched with Sn were similar to those results and trends reported by Kulaitienė et al. [45] in Stinging nettle-fortified yogurt bites and those by Öztürk-Yalçın et al. [15] on mint-fortified kefir ice cream. Both reflect the decrease in TPC and antioxidant activity during storage and the promoted the positive effects on prebiotics. The decrease in antioxidant activity and TPC of the samples during storage might be due to the degradation of polyphenols by the probiotic bacteria in kefir and the production of aromatic acids such as phenyl propanoic acid, acetic acid, and benzoic acid during refrigeration [46]. The Stinging nettle prebiotic effects may be attributed to its own antioxidant potential, including its rich phenolic compounds and vitamin A and vitamin C content, all potent ingredients found in abundance in nettle powder [47]. Furthermore, a study by Bhanja Dey et al. [48] explained that although probiotics hydrolyze polyphenols to some extent, they also produce certain enzymes that can support the release of plant polyphenols from their matrix.

4.4. Microbial Survival After In Vitro Digestion and Colonic Fermentation of Kefir

The dramatic decrease in probiotic activity in kefir samples after undergoing in vitro gastric digestion might be due to a combination of the extremely low pH (~3.0) and the antimicrobial properties of pepsin [49,50]. The viability of LAB after in vitro digestion in the present study was similar to the results of Kaur Sidhu et al. [19] using chickpea-fortified yogurt after 2 h of gastric and small intestinal digestion, which were in the range of 3–5 log CFU/g. The protective effect of Sn on the in vitro digestion of kefir LAB may originate from the binding effect of some of its proteins and dietary fibers on bile salts, as well as the physical protection of prebiotics [51,52]. However, adding Sn at 0.25% and 0.5% did not produce significant (p > 0.05) differences, which is also consistent with the study by Kaur Sidhu et al. [19]. The positive effects of Sn on probiotics during colonic fermentation may be attributed to the alteration of the composition and activity of the intestinal microflora by the phenolic compounds and dietary fiber in Sn. It was reported by Boto-Ordóñez et al. [53] that phenolic compounds and dietary fiber, in general, can promote the growth of probiotics, while inhibiting the growth of pathogenic bacteria. These observations were supported by the findings of Zahid et al. [17], who reported a significant increase in the Lactobacillus and total plate counts in yoghurt enriched with mango peel as a source of fiber and phenolic compounds during in vitro fermentation. Furthermore, similar to the results reported in kefir samples subjected to in vitro fermentation after 1 day of refrigerated storage, there were insignificant (p > 0.05) differences between samples treated with 0.25% or 0.5% Sn. Such results confirmed that adding Sn to kefir at 0.25% will be good enough to protect the survival of lactic acid bacteria counts represented by Lactobacillus sp.

4.5. Short-Chain Fatty Acid (SCFA) Production After In Vitro Colonic Fermentation of Kefir

The concentration of SCFAs released in the colon is affected by several conditions, including intestinal functioning time, host diet composition, and microflora [54]. The main types of SCFAs detected in kefir enriched with Sn and the differences in their quantities were similar to those reported by Zahid et al. [17] using mango peel yogurt, with acetic acid being the most abundant fatty acid, followed by butyric acid and propionic acid. However, different from the trend in this study, some studies showed a significant decrease in acetic and propionic acids after 72 h of fermentation [27]. While the trend of higher butyric acid production at 72 h compared to 48 h was consistent with the results of some studies [23], such differences may be due to differences in the composition of the probiotic flora [55]. The promoting effect of Sn on the production of SCFAs in kefir yogurt could be attributed to Sn phenolic and dietary fiber effects. Dietary fiber can better reach the colon as a substrate for the fermentation of intestinal microflora and modulate the intestinal microflora by increasing the number of probiotics such as Lactobacillus and Lactococcus spp., and in this way promote the production of SCFAs [56]. The abundant isoflavones in Sn may be degraded by microorganisms into small molecules of phenolic substances such as phenyl-valeric acid and phenyl-acetic acid, leading to the increase in SCFA content [57]. This mechanism has been demonstrated by citrus flavanones, which can traverse the intestinal tract to reach the colon and be degraded by intestinal microorganisms into phenolic metabolites that increase the content of SCFAs [58].

5. Conclusions

The overall aim of this study was to explore the potential enhancement of the nutritional value and the physicochemical properties of kefir by the addition of Stinging nettle (Sn). The results showed that the use of Sn powder had a favorable prebiotic effect and was effective in increasing the activity of Lactobacillus and Lactococcus spp. in kefir during the refrigerated storage, as well as in increasing the antioxidant activity and the total polyphenol content of the kefir. In addition, adding Sn enhanced the viability of intestinal microflora during in vitro colonic fermentation. Adding Sn increased the LAB counts and improved the production of SCFAs after colonic fermentation. These findings represent the novelty of this current study. Furthermore, the addition of Sn also affected the physicochemical properties of kefir yogurt and increased the pH, titratable acidity, viscosity and syneresis of kefir. However, adding Sn affected the color of the yoghurt and the kefir became darker and more yellow. The results showed also that adding Sn at 0.25% concentration yielded the most stable product quality with enhanced nutritional value.