Comprehensive Investigation of Humic-Mineral Substances from Oxyhumolite: Effects on Fatty Acid Composition and Health Lipid Indices in Milk and Cheese from Holstein-Friesian Cows

Abstract

The objective of this study was to determine the effect of the oxyhumolite supplementation in the feed of cows on the profile and the nutritional indices of fatty acids (FA) in milk and cheese. To the diet of 30 cows, a 100 g/per cow/per day humic-mineral supplement with 65% humic acids was included. Milk was sampled three times: control (0 day, before the dietary modification) and on days 30 and 60 after the introduction of the supplement. For chemical analyses, samples from each cow and for cheese manufacture samples of bulk milk were taken. Fat content in the milk and the cheese, and partial sums of fatty acids profile, nutritional fatty acid ratios and indices were determined. The indicated changes in milk fat quality are difficult to unequivocally assess from a dietary point of view. Negative changes were found in the increase in the proportion of hypercholesterolemic acids (HSFA), atherogenic (AI) and thrombogenic (TI) indices, and a decrease in the hypocholesterolemic/hypercholesterolemic (h/H) ratio, the proportion of desired (DFA) and monounsaturated (MUFA) FA over time of supplementation. Positive changes were also found: the increase in polyunsaturated (PUFA), branched-chain (BCFA), short- and medium-chain (SCSFA) and long-chain (LCSFA) FA percentages, and also content was reported. The cheese (30 and 60 d) showed an increase in the content (mg/100 g) of SFA (including SCFA and LCSFA), OCFA (odd-chain FA), BCFA, MUFA, and DFA. Adverse changes were observed with an increase in PUFA n6, the n6/n3 ratio, and HSFA and TI. No significant differences were found for the AI index. The preliminary results obtained are promising, although further research is needed.

1. Introduction

Milk and dairy products provide high-quality protein, micronutrients, and numerous bioactive compounds, and their introduction into the daily diet is highly recommended at a minimum of one serving. In most countries, the consumption of fat-free or low-fat dairy products is recommended due to the high saturated fatty acid (SFA) content in raw whole-cream milk [1]. Dairy products are estimated to provide 20% of saturated fat in the U.S. diet and 17–41% in European countries [2]. High intakes of SFAs and industrial sources of trans fatty acids (TFAs) have been linked to an increased risk of developing cardiovascular diseases, a leading cause of mortality worldwide. Dairy fat contains almost 400 different fatty acids (FAs), thus it is the most complex source of fat in the human diet. It also contains a number of unique FAs of a biological nature, such as short- (SCSFA) and medium-chain fatty acids (MCSFA), trans fatty acids, and odd and branched chain fatty acids (OBCFA). It is noteworthy that the recommended consumption of low- or fat-free products contributes to a reduction in the dietary supply of these valuable components [3]. Giosuè et al. [4] reported that consumption of up to 200 g/day of full-fat or low-fat dairy products is not associated with death from any cause or with the development of cardiovascular diseases. In the case of consumption of larger quantities of dairy products, the available data do not allow a clear link to be established.

The cows’ diet is the key to modifying the nutritional value of milk. Pasture-based feeding of cows has been shown to positively influence the fatty acid profile of milk [5,6,7]. However, on large-scale farms keeping cows with high production potential, animal feeding is usually mainly based on hay and maize silage year-round. This feeding system makes it possible to correctly balance the ration, avoid nutritional and management difficulties, and exploit the animals’ production potential. A number of feed components that are sources of unsaturated fatty acids (UFAs) have been tested to modify the fatty acid profile, including vegetable oils and oilseeds (flax, canola, soybean, sunflower), fish oil, and algae [8,9,10]. Supplementation of cows’ diets with such components resulted in a reduction in milk fat content or no effect on the content of the basic components, with a reduction in the proportion of SFA in milk fat and an increase in the proportion of MUFA (monounsaturated fatty acids).

One of the basic tenets of the European Green Deal is counteracting antibiotic resistance, which is currently one of the most serious problems facing humanity. Bearing in mind that the Farm to Table Strategy and Pollution Eradication Action Plan includes a target to reduce by 50% the total EU sales of antimicrobials to livestock by 2030, the European Parliament promotes the use of innovative feed additives to maintain and improve animal health, as well as prevent disease and the need for antimicrobials [11]. Prebiotics, probiotics, enzymes, organic acids, plant extracts, and humic compounds, among others, are used as feed additives. They are environmentally friendly and have a positive impact on animal health and the quality of the raw materials obtained from them.

Humic compounds are formed through the decomposition of plant and animal residues in the soil. They are a source of fulvic, humic and humic acids, and minerals [12]. Oxyhumolite (oxidized brown carbon) is rich in these components [13]. Humic acids exhibit multidirectional effects including anti-inflammatory, antibacterial and antiviral, immunomodulatory, as well as reducing oxidative stress [14]. In an earlier study, Teter et al. [15] showed that the dietary supplementation of cows with a humic-mineral feed additive increased the fat content of milk and had a positive effect on the applicability of milk for cheese production. Thus, it seems reasonable to know the possible influence of the supplementation used on the nutritional value of milk fat, both in the raw material produced and in the derived products. The objective of this study was to determine the effect of the supplementation of oxyhumolite with active humic-mineral substances in cow feed on the profile and nutritional indices of fatty acids in milk and cheese.

2. Materials and Methods

2.1. Animals, Diet, and Milk Sampling

The study was carried out on a farm with 170 Holstein-Friesian cows, which were kept in a free-stall barn and fed using the PMR (Partialy Mix Ration) system. The basal diet consisted of maize silage (63%), haylage (30%), rapeseed meal (1.8%), high protein concentrate (39% protein, Biofeed, 2%), soybean meal (2.1%), straw (0.6%), protect buffer (Biofeed, 0.3%), and chalk (0.2%). Fifty cows that were in lactation 1 or 2 between their 30th and 120th day (average 68 days) were pre-selected from the herd. The average cows body weight was 654 ± 47 kg and the daily milk yield was 34.68 ± 5.12 kg. Milking took place twice a day; i.e., at 6.00 a.m. and 6.00 p.m. From the original group of 50 cows, 30 were randomly selected, and milk samples were taken from the morning milking (day 0). HUMAC^® Natur AFM (100 g per cow/per day), containing 65% humic acids on a dry matter basis, was then included in the diet of the cows in this group. The humic-mineral additive was introduced into the basal ration and mixed in the feed wagon with the other ingredients. The ingredient content of the supplement is shown in

Table 1. Ingredient content of HUMAC^® Natur AFM feed additive (in dry matter).

Component	Content
Humic acids	65%
Fulvic acids	5%
Minerals:	mg/100 g:
Ca	4.228
Mg	0.511
Fe	1.905
Cu	1.5
Zn	3.7
Mn	14.2
Co	0.124
Se	0.167
V	4.21
Mo	0.27

Milk samples were taken on days 0, 30, and 60, after the introduction of the feed additive. The total study material consisted of 90 milk samples (30 animals × 3 samplings) individually taken from full milking from each cow during morning milking at 500 mL each. The fat content was determined in fresh milk and the fatty acid profile in frozen milk (−20 °C) up to 30 days after sampling. All analyses were performed in triplicate.

. The basal feed was estimated for 30 kg of milk yield. Per every 2.5 kg of milk above 30 kg, cows additionally received 1 kg of complete fed with 18% protein from a feed station.

2.2. Cheese Samples

Cheese was made under laboratory conditions from bulk milk collected three times during the experiment: before the introduction of the feed additive into the cows’ diet (day 0), on day 30, and 60 after the introduction of the feed additive. Four cheeses in the shape of a wheel were made from each batch of milk. The milk came from the morning milking and was not standardized for fat content. The milk was heat treated (65 °C ± 1 °C for 30 min), cooled to 32 °C, and CaCl₂ (0.02%) and starter cultures (2%) containing Lactococcus lactis subsp. lactis and Streptococcus thermophilus were added. Rennet, containing 80% chymosin and 20% pepsin (0.0513 IMCU/milk mL), was then added to the milk. All the additives used were manufactured by GAP, Food Additives, Nowy Sącz, Poland. The milk was thoroughly mixed, stabilized, and left for 40 min to coagulate. After the milk had clotted, the curd was cut with the harps into 1.5 × 1.5 × 1.5 cm cubes and left for the separation of whey (15 min). The cheese mass was then mixed and reheated to 36 °C to express the whey, and placed in molds. The cheese was pressed for 3 h at 20 °C, then cooled (6 °C). After 12 h, the cheese wheels were brined in brine (NaCl 18%) at 12–14 °C for 5 h and dried. They were kept refrigerated (5 °C) until the assays were performed.

2.3. Laboratory Analyses

2.3.1. Analyses of Milk

The fat content was determined in the milk samples using the Bentley 150, Infrared Milk Analyzer (Bentley Instruments, Inc., Chaska, MN, USA), which measures the energy absorption at specific wavelengths in the mid-infrared (MIR) region.

The fatty acid profile was determined, following fat extraction by the Röse Gottlieb method [16], after which direct conversion to fatty acid methyl esters (FAME) was performed by transmethylation of the fat sample using a mixture of concentrated H₂SO₄ (95%) and methanol according to the American Oil Chemists’ Society (AOCS) Official Method Ce 2-66 [17]. Gas chromatographic analyses were performed in detail according to Dopieralska et al. [18] using the Varian GC 3900 (Walnut Creek, CA, USA) gas chromatograph with a flame ionization detector (FID) and capillary column with a stationary phase of high polarity (100 m length × 0.25 mm I.D. × 0.25 μm film thickness; CP 7420 Agilent Technologies, Santa Clara, California, USA). Details of fatty acid identification are described in the paper [18]. The results of the measurements were compiled using Star GC Workstation v. 5.5. The analyses were carried out in triplicate. The FAME peak areas were corrected by FID response factors for each fatty acid previously determined using a reference standard (Supelco 37 Component FAME Mix CRM 47885 Supelco, Supelco Inc., Bellefonte, PA, USA) and then converted to fatty acid (FA) using the FAME-to-FA conversion factor known as the Sheppard factor [19]. FAs were expressed as a percentage of the sum of detected FA (% of total FA), or in gravimetric contents (mg/100 mL milk), using the conversion factor for milk and milk products (0.945) for the calculation of total FA from total lipids [20] and also milk density (g/mL).

2.3.2. Analyses of Cheese

The fat content (g/100 g) was determined according to the Van Gulik method [21].

The fatty acid profile was determined following fat extraction using the Schmid-Bondzynski-Ratzlaff method [22]. The solvent was removed using a rotatory evaporator at 40 °C under reduced pressure. The extract was then dried at 40 °C under a stream of nitrogen, followed by frozen at −45 °C (LT U250, Nordic Lab, Vaerloese, Denmark). Conversion to fatty acid methyl esters (FAME) and GC analysis was performed according to the method described for milk samples.

2.4. Statistical Analysis

Data were subjected to one-way ANOVA using Dell Statistica ver. 13 software [23]. An individual cow was the experimental unit, and the main effect of the cow group, i.e., the control (0 d) and experimental diet with oxyhumolite (30 d and 60 d) on the fatty acid characteristics of milk and cheese, was analyzed. The Tukey’s test was used to determine the differences between means and considered significant at p < 0.05. In tables the mean ± standard deviation is presented. The main effect of the cow group, i.e., the control (0 d) and experimental diet with oxyhumolite (30 d and 60 d) on the fatty acid characteristics of milk and cheese, was analyzed assuming an individual cow as the experimental unit. In order to determine the differences between means the Tukey’s test was applied and indicated when p < 0.05.

3. Results and Discussion

3.1. Fat Content in Milk and Cheese

Cows’ milk fat content was shown to be higher after the introduction of the humic-mineral supplement from oxyhumolite in the diet, with significant changes (p < 0.05) found 60 days after the dietary modification (

Table 2. Fat content of milk and cheese made from it [Mean ± SD].

Item	Milk			Cheese
	0	30	60	0	30	60
Fat content (g/100 g)	3.58 b ± 0.56	3.72 b ± 0.63	3.97 a ± 0.54	21.50 b ± 0.49	22.88 b ± 0.43	25.31 a ± 1.44

0, 30, 60—days of dietary supplementation; ^{a, b}—means in rows marked with different uppercase superscripts significantly differ at p < 0.05.

). The results obtained are consistent with those of a previous study by Teter et al. [15]. The increase in milk fat content may have been the result of better utilization of feed ingredients under the influence of humic acids. Potůčková and Kouřimská [24] reported that humic compounds have the ability to modify the composition of the intestinal flora and thus improve the utilization of feed nutrients, which has a positive effect on the chemical composition of milk. The higher fat content of the milk translated into higher fat content in the produced cheese (Table 2). The cheese made from bulk milk 60 days after the introduction of the humic-mineral additive into the cows’ rations contained significantly (p < 0.05) more fat compared to the cheese made from the milk before the dietary modification (by 17.7%), as well as from the milk of cows fed with oxyhumolite for 30 days (by 10.6%)

3.2. Profile and Content of Fatty Acids in Milk

It has been demonstrated that the supplementation of oxyhumolite in the cows’ feed significantly affected the fatty acid profile and health lipid indices in milk and the cheese made from it. There was a significantly (p < 0.05) higher proportion of SFA, PUFA (polyunsaturated fatty acids), and those with a hypercholesterolaemic effect (HSFA). In turn, a lower proportion of MUFA and desirable fatty acids (DFA), and lower values of the h/H (hypocholesterolaemic/hypercholesterolaemic) index, while higher levels of the AI (atherogenic) and TI (thrombogenic) indices of milk fat, were found (

Table 3. Effect of supplemental humic-mineral substances in cows’ diets on partial sums of fatty acids, nutritional fatty acid ratios, and indices in milk [Mean ± SD].

Fatty Acids	% of Total FA			mg/100 mL
	0	30	60	0	30	60
SCSFA	12.84 b ± 1.49	12.52 b ± 0.50	14.10 a ± 1,49	395.87 b ± 58.76	429.64 ab ± 79.63	467.53 a ± 37.83
LCSFA	52.50 b ± 1.14	54.02 a ± 1.42	55.15 a ± 1.35	1792.76 ± 306.84	1954.65 ± 435.17	1919.66 ± 167.03
SFA	65.33 b ± 1.89	66.54 b ± 1.79	69.26 a ± 2.19	2188.63 ± 348.04	2384.29 ± 514.88	2387.18 ± 175.03
OCFA	2.46 ± 0.28	2.40 ± 0.10	2.27 ± 0.16	83.79 ± 15.26	86.43 ± 17.95	79.18 ± 9.91
BCFA	2.27 b ± 0.18	2.57 a ± 0.11	2.58 a ± 0.22	76.74 b ± 6.21	91.87 a ± 14.29	89.70 a ± 8.93
MUFA	28.12 a ± 1.41	26.50 ab ± 1.49	24.89 b ± 1.97	1015.21 ± 216.94	995.34 ± 266.03	871.80 ± 149.61
MUFA trans	1.88 b ± 0.20	2.18 ab ± 0.31	2.24 a ± 0.46	69.89 ± 16.48	86.52 ± 25.02	76.48 ± 17.33
MUFA cis	26.24 a ± 1.46	24.32 b ± 1.20	22.63 c ± 1.86	940.72 ± 202.55	908.81 ± 241.18	795.32 ± 140.25
PUFA	3.05 b ± 0.28	3.38 a ± 0.36	3.32 a ± 0.39	110.81 ± 20.32	131.18 ± 31.29	114.77 ± 20.35
n3	0.35 ± 0.09	0.37 ± 0.05	0.38 ± 0.08	13.86 ± 5.23	14.57 ± 4.20	13.15 ± 3.32
n6	1.42 ± 0.18	1.45 ± 0.15	1.55 ± 0.13	49.25 ± 6.07	55.38 ± 11.37	52.82 ± 7.16
n6/n3	4.21 ± 0.85	3.91 ± 0.38	4.23 ± 1.04	3.90 ± 1.06	3.88 ± 0.45	4.19 ± 0.96
PUFA/SFA	0.05 ± 0.003	0.05 ± 0.006	0.05 ± 0.006	0.05 ± 0.01	0.06 ± 0.01	0.05 ± 0.01
DFA	40.89 a ± 1.80	39.17 ab ± 2.98	37.43 b ± 2.39	1475.73 ± 306.38	1493.97 ± 408.02	1304.54 ± 208.78
HSFA	44.91 b ± 1.525	46.67 ab ± 2.84	47.40 a ± 1.90	1465.70 b ± 260.39	1616.94 ab ± 326.54	1657.23 a ± 122.35
h/H	0.51 a ± 0.04	0.45 ab ± 0.05	0.42 b ± 0.05	0.58 a ± 0.04	0.49 ab ± 0.05	0.42 b ± 0.05
AI	2.42 b ± 0.51	2.66 ab ± 0.22	3.15 a ± 0.34	2.41 b ± 0.51	2.65 ab ± 0.22	3.14 a ± 0.33
TI	3.01 b ± 0.48	3.27 ab ± 0.15	3.68 a ± 0.32	3.05 b ± 0.49	3.31 ab ± 0.14	3.71 a ± 0.33

0, 30, 60—days of dietary supplementation; ^{a, b, c}—means in rows marked with different uppercase superscripts significantly differ at p < 0.05; SCSFA—short- and medium-chain saturated FAs: sum of C4:0, C6:0, C8:0, C10:0, C11:0 and C12:0; LCSFA—long-chain saturated FAs: sum of C13:0, C14:0, C15:0, C16:0, C17:0, C18:0, C20:0, C21:0, C22:0, C23:0 and C24:0; SFA—saturated FAs, (SCSFA + LCSFA); OCFA—odd-chain FAs; sum of C11:0, C13:0, C15:0, C21:0, C23:0, C17:0 and C17:1c9; BCFA—branched-chain FAs, sum of C13:0i, C13:0a, C14:0i, C15:0i, C15:0a, C16:0i, C17:0a, C17:0i and C18:0i (i—iso, a—anteiso); MUFA trans—monounsaturated FAs, sum of C18:1t6/7, C18:1t9, C18:1t10, C18:1t11, C18:1t15 and C18:1t16; MUFA cis—monounsaturated FAs, sum of C10:1, C14:1c9, C16:1c13, C16:1c7, C16:1c9, C17:1c9, C18:1c9, C18:1c11, C18:1c12 and C20:1c11; PUFA—polyunsaturated FAs, sum of C18:2trans, C18:2t9t12, C18:2t9c12, C18:2c9t12, C18:2n6, C18:2cis, C18:3n3, ΣC18:3trans, ΣCLA, C20:3n6, C20:4n6, C20:5n3 and C22:5n3; DFA—desirable fatty acids—(MUFA + PUFA + C18:0); h/H—hypocholesterolaemic/hypercholesterolaemic ratio—(C18:1c9 + C18:2n6 + C20:4n6 + C20:5n3 + C22:5n3) ÷ (C12:0 + C14:0 + C16:0), HSFA—hypercholesterolaemic saturated fatty acids-(C12:0 + C14:0 + C16:0); AI—atherogenic index = (C12:0 + 4 × C14:0 + C16:0) ÷ (MUFA + PUFA) and TI—thrombogenic index = (C14:0 + C16:0 + C18:0) ÷ (0.5 × MUFA + 0.5 × n6 + 3 × n3 + n3 ÷ n6).

).

Dairy fat contains on average 70% SFA, 25% MUFA, and 5% PUFA, while the ideal FA profile, from a human health perspective, should be 8% SFA, 82% MUFA, and 10% PUFA [25]. One strategy to prevent people from developing chronic non-communicable diseases is to change the fatty acid composition of their diet. This approach is based on reducing the intake of SFAs, increasing the intake of MUFAs and PUFAs, and especially changing the ratio of n6/n3 fatty acids [26].

In the saturated fatty acids group, there was a higher proportion of short- and medium-chain (SCSFA), and long-chain (LCSFA) acids. There was also a significantly higher proportion of branched-chain acids (BCFA). However, when the results were expressed in an absolute amount (mg/100 mL milk), significant differences were only observed for SCSFA and BCFA. The detailed fatty acid profile of milk fat is shown in

Table 4. Effect of supplemental humic-mineral substances in cows’ diets on fatty acid composition of milk [Mean ± SD].

Fatty Acid	% of Total FA			mg/100 mL
	0	30	60	0	30	60
C4:0	3.22 ± 0.31	3.20 ± 0.04	3.24 ± 0.18	100.14 ± 17.40	105.65 ± 25.23	102.67 ± 18.70
C6:0	2.19 ± 0.22	2.13 ± 0.10	2.29 ± 0.15	68.04 ± 9.87	72.09 ± 13.26	74.72 ± 13.72
C8:0	1.27 ± 0.17	1.23 ± 0.09	1.38 ± 0.14	39.26 b ± 6.28	42.37 ab ± 6.70	45.88 a ± 4.40
C10:0	2.85 ab ± 0.45	2.77 b ± 0.20	3.27 a ± 0.49	87.17 b ± 16.16	96.71 ab ± 15.34	110.47 a ± 12.94
C10:1	0.30 ± 0.06	0.29 ± 0.05	0.34 ± 0.04	9.53 b ± 1.81	10.27 ab ± 0.87	11.71 a ± 1.07
C11:0	0.07 ± 0.02	0.06 ± 0.01	0.08 ± 0.03	2.31 ± 0.97	2.34 ± 0.81	2.85 ± 0.84
C12:0	3.24 b ± 0.54	3.13 b ± 0.20	3.84 a ± 0.64	99.14 b ± 19.76	110.49 ab ± 18.42	130.93 a ± 18.08
C13:0 iso	0.08 b ± 0.01	0.07 b ± 0.01	0.10 a ± 0.02	2.55 b ± 0.42	2.46 b ± 0.39	3.45 a ± 0.59
C13:0 anteiso	0.12 ± 0.03	0.12 ± 0.02	0.13 ± 0.02	3.77 ± 1.30	4.16 ± 0.97	4.59 ± 0.93
C13:0	0.13 ± 0.02	0.13 ± 0.02	0.13 ± 0.03	4.01 ± 1.08	4.57 ± 0.35	4.58 ± 0.73
C14:0 iso	0.11 b ± 0.03	0.14 a ± 0.02	0.15 a ± 0.04	3.87 b ± 1.00	5.10 ab ± 0.75	5.33 a ± 1.27
C14:0	10.98 b ± 1.10	10.90 b ± 0.75	12.03 a ± 0.80	349.85 b ± 53.59	387.99 ab ± 63.08	414.72 a ± 17.02
C15:0 iso	0.23 b ± 0.02	0.29 a ± 0.01	0.26 a ± 0.03	7.83 b ± 1.14	10.52 a ± 2.31	9.21 a ± 1.23
C14:1c9	0.95 ± 0.14	0.98 ± 0.06	1.09 ± 0.12	30.24 b ± 6.42	33.88 ab ± 5.57	37.60 a ± 4.53
C15:0 anteiso	0.47 b ± 0.04	0.53 a ± 0.02	0.55 a ± 0.04	15.46 b ± 2.02	18.99 a ± 3.69	19.04 a ± 2.09
C15:0	1.13 ± 0.06	1.19 ± 0.09	1.12 ± 0.09	36.85 ± 7.18	42.54 ± 6.59	39.14 ± 4.78
C16:0 iso	0.28 b ± 0.08	0.40 a ± 0.11	0.37 a ± 0.07	2.55 b ± 0.42	2.46 b ± 0.38	3.45 a ± 0.59
C16:0	30.70 ± 1.48	30.93 ± 1.33	31.92 ± 1.59	1016.71 ± 207.47	1118.47 ± 245.04	1111.59 ± 112.80
C16:1c13	0.06 ± 0.02	0.05 ± 0.01	0.06 ± 0.01	2.11 ± 0.65	1.90 ± 0.67	1.95 ± 0.39
C16:1c7	0.25 a ± 0.03	0.23 ab ± 0.02	0.21 b ± 0.03	8.74 a ± 1.89	8.37 ab ± 2.12	7.26 b ± 1.44
C16:1c9	1.61 ± 0.27	1.54 ± 0.08	1.42 ± 0.26	55.74 ± 16.64	56.45 ± 15.79	49.91 ± 13.15
C17:0 iso	0.33 ± 0.03	0.33 ± 0.03	0.32 ± 0.03	11.41 ± 1.41	11.96 ± 3.19	11.26 ± 1.69
C17:0anteiso	0.58 b ± 0.05	0.61 ab ± 0.04	0.63 a ± 0.05	19.42 b ± 1.47	21.98 a ± 3.75	21.91 a ± 1.69
C17:0	0.73 a ± 0.04	0.75 a ± 0.04	0.68 b ± 0.06	26.28 ± 5.38	27.31 ± 7.46	23.96 ± 4.10
C17:1c9	0.26 a ± 0.04	0.26 a ± 0.03	0.21 b ± 0.04	10.08 a ± 3.45	9.67 ab ± 3.02	7.51 b ± 2.05
C18:0 iso	0.08 ± 0.02	0.08 ± 0.03	0.07 ± 0.01	2.95 ± 1.56	2.88 ± 0.83	2.59 ± 0.43
C18:0	9.72 a ± 0.80	9.95 a ± 0.76	9.04 b ± 0.67	349.72 ± 75.12	367.46 ± 110.87	317.97 ± 47.38
C18:1t6/7	0.21 c ± 0.02	0.25 a ± 0.02	0.23 b ± 0.02	7.21 b ± 1.05	9.17 a ± 2.49	7.92 ab ± 1.44
C18:1t9	0.17 b ± 0.01	0.20 a ± 0.02	0.18 b ± 0.02	6.11 ± 1.02	7.26 ± 1.69	6.19 ± 0.84
C18:1t10	0.26 b ± 0.02	0.33 a ± 0.02	0.30 a ± 0.04	9.27 b ± 1.74	12.04 a ± 3.14	10.32 ab ± 1.93
C18:1t11	0.71 b ± 0.11	0.93 a ± 0.05	0.81 a ± 0.12	28.06 ± 9.99	33.81 ± 8.05	28.12 ± 5.01
C18:1c9	21.52 a ± 1.35	20.51 a ± 0.86	18.40 b ± 1.55	784.83 a ± 180.50	748.14 ab ± 201.31	645.02 b ± 118.75
C18:1t15	0.23 ± 0.10	0.34 ± 0.11	0.35 ± 0.15	7.56 ± 3.18	13.42 ± 6.40	12.20 ± 14.03
C18:1c11	0.94 a ± 0.09	0.82 b ± 0.04	0.74 b ± 0.11	32.40 a ± 6.54	29.83 ab ± 8.04	26.09 b ± 5.44
C18:1c12	0.23 b ± 0.02	0.29 a ± 0.06	0.26 ab ± 0.06	7.91 b ± 1.14	10.78 a ± 4.37	9.08 ab ± 1.96
C18:1c13	0.15 ± 0.08	nd.	0.12 ± 0.01	5.01 ± 0.35	nd.	5.28 ± 0.42
C18:1t16	0.32 b ± 0.03	0.40 a ± 0.09	0.33 b ± 0.02	11.68 ab ± 2.82	14.18 a ± 3.06	11.36 b ± 1.72
C18:2 trans	0.14 ± 0.01	nd.	0.10 ± 0.02	3.12 ± 0.21	nd.	3.27 ± 0.32
C18:2c9t12	0.27 ± 0.07	0.31 ± 0.05	0.32 ± 0.08	9.38 ± 3.36	11.77 ± 4.39	11.26 ± 3.09
C18:2t9t12	0.27 b ± 0.07	0.38 a ± 0.06	0.29 b ± 0.05	9.89 b ± 2.79	13.84 a ± 3.25	10.31 b ± 2.45
C18:2t9c12	0.09 b ± 0.04	0.20 a ± 0.04	0.13 ab ± 0.08	3.69 b ± 2.00	7.51 a ± 3.06	4.68 ab ± 3.01
C18:2n6	1.27 ± 0.10	1.32 ± 0.05	1.33 ± 0.12	45.05 ± 5.98	48.18 ± 10.55	46.46 ± 5.82
C18:2 cis	0.12 ± 0.04	0.15 ± 0.04	0.11 ± 0.06	4.21 ± 1.88	5.47 ± 2.42	4.03 ± 2.56
ΣC18:3 trans	0.13 ± 0.04	0.11 ± 0.03	0.13 ± 0.04	4.50 ± 1.46	4.24 ± 2.26	4.84 ± 1.72
C18:3n3 ALA	0.30 ± 0.07	0.31 ± 0.04	0.33 ± 0.04	11.27 ± 3.46	11.60 ± 3.71	11.49 ± 2.05
C20:0	0.14 ± 0.03	0.15 ± 0.05	0.14 ± 0.03	4.69 ± 1.26	5.82 ± 3.09	5.00 ± 1.46
ΣCLA	0.40 b ± 0.05	0.52 a ± 0.07	0.44 ab ± 0.13	15.19 ± 4.25	18.40 ± 3.13	15.30 ± 4.51
C20:1c11	0.07 ± 0.01	nd.	0.06 ± 0.05	2.33 ± 0.45	nd.	2.13 ± 0.37
C21:0	0.22 ± 0.08	nd.	0.11 ± 0.02	7.33 ± 2.79	nd.	3.59 ± 0.46
C20:3n6	0.09 ± 0.01	0.11 ± 0.02	0.10 ± 0.03	2.85 ± 0.45	3.68 ± 0.35	3.62 ± 1.03
C20:4n6	0.12 ± 0.03	0.12 ± 0.03	0.11 ± 0.03	4.02 ± 0.83	4.44 ± 1.94	3.77 ± 1.25
C22:0	0.07 ± 0.02	0.07 ± 0.02	0.09 ± 0.02	2.52 ± 1.24	1.98 ± 0.76	3.04 ± 1.37
C23:0	0.60 a ± 0.03	nd.	0.08 b ± 0.02	19.46 a ± 0.99	nd.	2.86 b ± 0.91
C24:0	nd.	nd.	0.07 ± 0.01	nd.	nd.	2.29 ± 0.23
C22:5n3	0.09 ± 0.02	0.08 ± 0.01	0.08 ± 0.02	3.00 ± 0.77	2.97 ± 0.62	2.82 ± 0.62

0, 30, 60—days of dietary supplementation; nd.—not detected; ∑CLA—conjugated linoleic acid: sum of 18:2 c9,t11, 18:2 t9,c11, 18:2 c7,t9, 18:2 t7,c9, 18:2 c11,t13, 18:2 t11,c13; ^{a, b, c}—means in rows marked with different uppercase superscripts differ significantly at p < 0.05.

.

Limiting the supply of SFAs, particularly C12:0, C14:0, and C16:0 acids, as well as industrial sources of TFAs, is a key dietary recommendation to reduce the risk of cardiovascular disease [27]. The American Heart Association and the American College of Cardiology recommend limiting energy from saturated fatty acids to <7% of total energy intake [28]. In contrast, according to the European Food Safety Authority (EFSA) [29], saturated fatty acids are synthesized by the body and are not required in the diet, hence their supply should be as low as possible. It should be emphasized that the presence of SCSFAs is a unique characteristic of milk fat. They are formed by the rumen microflora of ruminants and are not found in other fats. In the human body, these acids have a number of important biological functions and are the source of, among other things, the energy necessary for the functioning of the heart, liver, kidneys, nervous system, and muscles. C4:0, C6:0, C8:0, and C10:0 acids are oxidized to acetyl-CoA in the liver and do not affect the activity of low-density lipoprotein receptors. They do not increase blood lipid levels, and thus, do not pose a risk of developing obesity and cardiovascular diseases [30]. Dairy products are an important source of calcium, which interacts with SFA to form calcium-FA soaps, thereby increasing fecal fat excretion, which improves the HDL to LDL cholesterol ratio [31].

The reduction in the proportion of MUFA and C18:0 acids resulted in a significant decrease in the proportion of DFA. In the MUFA profile, there was a significantly (p < 0.05) higher proportion of trans-configuration acids and a lower proportion of cis-configuration acids. With regard to the content of these fatty acids in milk, however, the differences found were not statistically significant. Trans fatty acids are negatively perceived by consumers, but a distinction must be made between trans fatty acids of natural origin and those industrially obtained. Among the trans isomers found in milk fat, vaccenic acid (isomer C18:1 t-11) is particularly important. It exhibits antiatherosclerotic effects by lowering blood levels of triglycerides and LDL cholesterol fractions, anti-cancer effects, and beneficial effects on the immune system. Through desaturation, conjugated linoleic acid (CLA) is formed from vaccenic acid [32,33]. The effect of introducing the humic-mineral substances into the cows’ diets in the present study was a significantly higher proportion of the C18:1 t-11 isomer in milk fat (Table 4). The highest proportion of PUFAs (3.38% FA) (Table 3) was found in milk fat at 30 days of supplementation, which was mainly due to the proportion of C18:2 t9t12 and C18:2 t9c12 acids (Table 4).

Health-related indices such as n6/n3, PUFA/SFA, h/H, HSFA, AI, and TI indices are used to assess the nutritional value and potential impact of product consumption on consumer health. A ratio of PUFA/SFA in the diet above 0.45 and a n6/n3 below 4.0 reduces the risk of developing cardiovascular diseases and cancer [34]. In our study, the PUFA/SFA ratio in milk fat ranged from 0.04 to 0.05 and n6/n3 from 3.91 to 4.23. Such a low value for the PUFA/SFA ratio was due to the high content of SFA acids. There was no significant effect of the use of humic-mineral substances in the feeding of cows on the proportion of these acids. However, significant (p < 0.05) changes were found in the values of the HSFA, h/H, AI, and TI indices, resulting from a higher proportion of hypercholesterolemic fatty acids and thus a deterioration in the nutritional quality of milk fat after the introduction of the dietary modification in cows (Table 3). Ulbricht and Southgate [35] proposed the AI and TI indices. As the values of these indices increase, the risk of cardiovascular disease increases, so products with the lowest possible levels of AI and TI should be selected [36]. In our study, after 60 days of supplementation, the AI value of milk fat was 30% higher and the TI value was 22% higher, but the index values were not higher than 4. The ranges of AI values for cereals, fish, meat, and dairy products are 0.084–0.55, 0.21–1.41, 0.165–1.32, and 1.42–5.13, respectively, and the TI index is 0.139–0.56, 0.14–0.87, 0.288–1.694, and 0.39–5.04, respectively. It is recommended to consume products with the lowest possible AI and TI values to minimize the risk of developing cardiovascular disease, but current dietary guidelines do not recommend values for these indices [36].

To the best of the authors’ knowledge, only two papers have so far been published on the effect of using humic substances in the feeding of cows on the fatty acid profile of milk. The results of the presented studies do not correspond with previous reports. Hassan et al. [37] fed clay-derived humic substances to cows at two doses: 5 and 10 g/kg feed (no information on the content of humic acids). Regardless of the dose used, there was no change in the fat content of the milk. There were also no significant changes in the proportion of SCSFA and MUFA. However, there was an increase in the proportion of PUFA, including isomeric forms of linoleic acid (C18:2t9t12 and C18:2c9t12), and a decrease in the values of the AI and TI indices and the PUFA/SFA ratio. Kholif et al. [38] fed humic substances to cows at doses of 20 and 40 g per cow per day. The feed additive used contained 90% humic acids and 10% minerals. Beneficial changes in the fatty acid profile were shown; i.e., a significant increase in the proportion of MUFA, PUFA, and CLA, and a reduction in AI values from 2.32 to 1.97 with the higher dose of the supplement applied. No changes were found in the proportion of SFA. The ambiguity of the information presented may be due to the use of humic substances of different origins, and therefore chemical composition or dosage, in the study. This can be a problem in comparing the results of studies on humic substances as animal supplements, so further research is needed to expand knowledge in this area.

3.3. Profile and Fatty Acid Content of Cheese

The detailed fatty acid profile and their ratios and indices in the cheese are shown in

Table 5. Effect of supplemental humic-mineral substances in cows’ diets on partial sums of fatty acids, nutritional fatty acid ratios, and indices in cheese [Mean ± SD].

Fatty Acids	% of Total FA			mg/100 g
	0	30	60	0	30	60
SCSFA	12.00 b ± 0.06	12.09 a ± 0.08	11.92 b ± 0.07	2345.08 c ± 11.57	2512.26 b ± 62.78	2736.41 a ± 149.40
LCSFA	53.98 ± 0.23	54.27 ± 0.46	53.96 ± 0.27	11051.52 c ± 101.89	11822.56 b ± 299.70	12971.08 a ± 254.16
SFA	65.98 ± 0.14	66.36 ± 0.56	65.88 ± 0.29	13396.60 c ± 170.20	14334.81 b ± 399.46	15707.49 a ± 880.71
OCFA	2.37 b ± 0.05	2.41 a ± 0.03	2.40 a ± 0.04	484.82 c ± 10.16	524.30 b ± 7.95	566.80 a ± 15.28
BCFA	2.42 ± 0.10	2.32 ± 0.07	2.36 ± 0.09	495.14 b ± 7.17	504.17 b ± 9.24	566.77 a ± 31.27
MUFA	28.06 ab ± 0.03	27.76 b ± 0.35	28.51 a ± 0.22	5742.62 b ± 5.13	6043.86 b ± 39.37	6851.40 a ± 406.69
MUFA trans	2.35 ± 0.03	2.39 ± 0.09	2.34 ± 0.13	480.35 ± 4.72	505.58 ± 19.60	538.05 ± 41.53
MUFA cis	25.71 ab ± 0.02	25.37 b ± 0.34	26.15 a ± 0.14	5262.27 b ± 0.41	5538.02 b ± 34.68	6313.25 a ± 369.29
PUFA	3.53 ± 0.11	3.56 ± 0.21	3.25 ± 0.18	726.98 ± 22.87	778.71 ± 33.46	787.81 ± 87.16
n3	0.55 a ± 0.04	0.44 ab ± 0.08	0.39 b ± 0.08	112.34 ± 8.87	97.13 ± 13.42	94.25 ± 19.84
n6	1.45 ± 0.05	1.47 ± 0.11	1.49 ± 0.06	299.29 b ± 10.55	322.33 ab ± 18.28	360.81 a ± 35.72
n6/n3	2.68 b ± 0.30	3.35 ab ± 0.34	3.94 a ± 0.86	2.68 b ± 0.31	3.35 ab ± 0.34	3.94 a ± 0.86
PUFA/SFA	0.05 ± 0.001	0.05 ± 0.003	0.05 ± 0.001	0.05 ± 0.001	0.05 ± 0.001	0.05 ± 0.001
DFA	42.26 ± 0.12	41.69 ± 0.74	41.63 ± 0.21	8666.97 b ± 24.50	9093.57 b ± 25.06	10024.98 a ± 609.82
HSFA	44.05 ± 0.12	44.68 ± 0.72	44.95 ± 0.20	8996.74 b ± 23.54	9711.22 b ± 340.77	10780.65 a ± 597.13
h/H	0.52 ± 0.3	0.50 ± 0.02	0.51 ± 0.03	0.52 ± 0.001	0.50 ± 0.020	0.51 ± 0.003
AI	2.49 ± 0.02	2.56 ± 0.11	2.51 ± 0.10	2.48 ± 0.13	2.55 ± 0.19	2.50 ± 0.21
TI	3.10 b ± 0.04	3.21 a ± 0.11	3.17 ab ± 0.05	3.14 b ± 0.03	3.26 a ± 0.13	3.19 ab ± 0.05

0, 30, 60—days of dietary supplementation; ^{a, b, c}—means in rows marked with different uppercase superscripts differ significantly at p < 0.05; SCSFA—short- and medium-chain saturated FAs: sum of C4:0, C6:0, C8:0, C10:0, C11:0 and C12:0; LCSFA—long-chain saturated FAs: sum of C13:0, C14:0, C15:0, C16:0, C17:0, C18:0, C20:0, C21:0, C22:0, C23:0 and C24:0; SFA—saturated FAs, (SCSFA + LCSFA); OCFA—odd-chain FAs; sum of C11:0, C13:0, C15:0, C21:0, C23:0 C17:0 and C17:1c9; BCFA—branched-chain FAs, sum of C13:0i, C13:0a, C14:0i, C15:0i, C15:0a, C16:0i, C17:0a, C17:0i and C18:0i (i—iso, a—anteiso); MUFA trans—monounsaturated FAs, sum of C18:1t6/7, C18:1t9, C18:1t10, C18:1t11, C18:1t15 and C18:1t16; MUFA cis—monounsaturated FAs, sum of C10:1, C14:1c9, C16:1c13, C16:1c7, C16:1c9, C17:1c9, C18:1c9, C18:1c11, C18:1c12 and C20:1c11; PUFA—polyunsaturated FAs, sum of C18:2trans, C18:2t9t12, C18:2t9c12, C18:2c9t12, C18:2n6, C18:2cis, C18:3n3, ΣC18:3trans, ΣCLA, C20:3n6, C20:4n6, C20:5n3 and C22:5n3; DFA—desirable fatty acids—(MUFA + PUFA + C18:0); h/H—hypocholesterolaemic/hypercholesterolaemic ratio—(C18:1c9 + C18:2n6 + C20:4n6 + C20:5n3 + C22:5n3) ÷ (C12:0 + C14:0 + C16:0), HSFA—hypercholesterolaemic saturated fatty acids-(C12:0 + C14:0 + C16:0); AI—atherogenic index = (C12:0 + 4 × C14:0 + C16:0) ÷ (MUFA + PUFA) and TI—thrombogenic index = (C14:0 + C16:0 + C18:0) ÷ (0.5 × MUFA + 0.5 × n6 + 3 × n3 + n3 ÷ n6).

and

Table 6. Effect of supplemental humic-mineral substances in cows’ diets on fatty acid composition of cheese (Mean ± SD).

Fatty Acids	% of Total FA			mg/100 g
	0	30	60	0	30	60
C4:0	3.16 a ± 0.01	3.20 a ± 0.03	3.06 b ± 0.03	589.27 b ± 12.41	634.40 ab ± 16.32	669.39 a ± 35.97
C6:0	2.01 b ± 0.01	2.06 a ± 0.02	1.98 b ± 0.02	387.18 b ± 11.96	423.00 ab ± 11.59	449.30 a ± 24.10
C8:0	1.15 ± 0.01	1.17 ± 0.02	1.15 ± 0.02	227.01 b ± 0.96	244.90 ab ± 7.65	265.43 a ± 17.42
C10:0	2.65 a ± 0.01	2.61 b ± 0.02	2.61 b ± 0.01	528.82 b ± 11.78	554.52 b ± 14.72	611.88 a ± 35.29
C10:1	0.25 ± 0.01	0.27 ± 0.02	0.27 ± 0.02	50.93 b ± 1.75	58.47 ab ± 3.91	64.13 a ± 4.47
C11:0	0.07 ± 0.02	0.06 ± 0.01	0.08 ± 0.02	14.73 ab ± 3.35	13.03 b ± 1.82	20.01 a ± 4.90
C12:0	2.96 b ± 0.03	2.99 b ± 0.02	3.04 a ± 0.02	598.07 b ± 5.93	642.40 b ± 13.44	720.39 a ± 38.26
C13:0 iso	0.08 ± 0.01	0.08 ± 0.01	0.08 ± 0.01	15.81 ± 0.66	16.94 ± 3.41	20.40 ± 1.98
C13:0 anteiso	0.12 ± 0.01	0.10 ± 0.02	0.12 ± 0.03	23.70 ± 0.57	21.14 ± 3.49	28.30 ± 5.88
C13:0	0.12 ± 0.01	0.12 ± 0.02	0.12 ± 0.03	24.19 ± 2.76	26.66 ± 4.61	28.71 ± 6.79
C14:0 iso	0.12 ± 0.02	0.12 ± 0.01	0.13 ± 0.02	25.43 ± 4.25	26.74 ± 0.37	31.37 ± 5.48
C14:0	10.28 ± 0.06	10.43 ± 0.13	10.40 ± 0.05	2092.86 b ± 22.50	2259.83 b ± 70.99	2486.11 a ± 139.57
C15:0 iso	0.26 ± 0.02	0.25 ± 0.02	0.24 ± 0.02	53.42 ± 1.01	54.91 ± 3.43	58.13 ± 6.33
C14:1c9	0.90 b ± 0.01	0.92 b ± 0.02	1.00 a ± 0.03	183.95 b ± 11.48	199.54 b ± 8.33	239.21 a ± 14.75
C15:0 anteiso	0.52 ± 0.01	0.50 ± 0.02	0.50 ± 0.05	105.56 ± 1.18	107.98 ± 5.36	119.84 ± 11.58
C15:0	1.14 ± 0.03	1.14 ± 0.04	1.11 ± 0.03	232.75 b ± 5.58	248.36 b ± 13.30	267.37 a ± 10.09
C16:0 iso	0.32 ± 0.02	0.29 ± 0.02	0.29 ± 0.04	65.24 ± 4.86	63.55 ± 3.84	69.51 ± 9.20
C16:0	30.81 ± 0.07	31.26 ± 0.59	31.51 ± 0.16	6305.81 b ± 15.11	6808.99 b ± 256.99	7574.14 a ± 419.86
C16:1c13	0.04 ± 0.02	0.06 ± 0.01	0.04 ± 0.01	7.70 ± 1.25	12.68 ± 0.35	10.71 ± 2.09
C16:1c7	0.23 ab ± 0.01	0.24 a ± 0.01	0.22 b ± 0.01	47.22 ± 0.14	51.77 ± 1.31	53.00 ± 4.24
C16:1c9	1.62 b ± 0.01	1.64 b ± 0.04	1.76 a ± 0.03	330.65 b ± 2.07	357.63 b ± 14.34	423.18 a ± 27.56
C17:0 iso	0.33 ± 0.01	0.32 ± 0.02	0.33 ± 0.02	67.80 ab ± 0.07	68.92 b ± 2.06	79.65 a ± 8.97
C17:0 anteiso	0.59 ± 0.02	0.57 ± 0.02	0.58 ± 0.02	120.29 b ± 3.35	124.55 b ± 1.91	139.33 a ± 4.66
C17:0	0.76 ± 0.02	0.75 ± 0.04	0.76 ± 0.05	155.67 ± 3.71	162.92 ± 7.46	182.63 ± 21.02
C17:1c9	0.28 ± 0.05	0.28 ± 0.01	0.28 ± 0.02	57.47 b ± 10.54	61.86 ab ± 3.73	68.09 a ± 1.78
C18:0 iso	0.09 ± 0.01	0.09 ± 0.03	0.08 ± 0.01	17.89 ± 1.98	19.46 ± 7.40	20.24 ± 3.07
C18:0	10.67 a ± 0.03	10.37 b ± 0.18	9.87 c ± 0.09	2197.37 b ± 6.75	2271.00 ab ± 4.40	2385.78 a ± 122.14
C18:1t6/7	0.26 ± 0.01	0.25 ± 0.04	0.26 ± 0.02	52.82 ± 0.63	54.16 ± 8.30	62.30 ± 5.71
C18:1t9	0.18 ± 0.01	0.19 ± 0.02	0.19 ± 0.02	36.66 b ± 1.80	41.21 ab ± 3.88	46.34 a ± 5.80
C18:1t10	0.30 ± 0.03	0.33 ± 0.02	0.29 ± 0.03	60.72 b ± 6.31	70.95 a ± 4.02	69.67 a ± 2.82
C18:1t11	0.97 a ± 0.01	0.90 a ± 0.04	0.83 b ± 0.04	197.66 ± 0.90	196.70 ± 6.49	200.76 ± 16.60
C18:1c9	21.33 ab ± 0.07	20.74 b ± 0.45	21.75 a ± 0.12	4365.10 b ± 15.24	4515.57 b ± 16.73	5148.02 a ± 316.73
C18:1t15	0.27 ± 0.01	0.27 ± 0.02	0.32 ± 0.06	55.68 ± 0.95	58.86 ± 5.37	76.29 ± 15.49
C18:1c11	0.85 b ± 0.02	0.85 b ± 0.02	0.89 a ± 0.02	174.72 b ± 3.31	186.11 b ± 7.40	213.87 a ± 13.22
C18:1c12	0.24 b ± 0.02	0.29 a ± 0.01	0.27 ab ± 0.02	48.38 b ± 4.22	62.71 a ± 2.89	64.30 a ± 3.79
C18:1c13	nd.	0.18 ± 0.01	0.16 ± 0.021	nd.	38.26 ± 3.12	37.80 ± 3.45
C18:1t16	0.38 ± 0.01	0.38 ± 0.02	0.34 ± 0.03	76.80 ± 2.18	83.70 ± 3.58	82.69 ± 9.72
C18:2 trans	0.08 ± 0.01	0.10 ± 0.02	0.09 ± 0.01	13.95 b ± 2.47	22.50 a ± 3.22	21.74 a ± 3.47
C18:2c9t12	0.40 a ± 0.05	0.37 a ± 0.03	0.29 b ± 0.04	82.44 ± 10.85	80.23 ± 7.17	69.31 ± 6.18
C18:2t9t12	0.27 ± 0.15	0.35 ± 0.02	0.30 ± 0.05	54.73 ± 30.85	76.14 ± 4.77	73.82 ± 13.70
C18:2t9c12	0.13 ± 0.02	0.15 ± 0.07	0.11 ± 0.03	27.44 ± 4.12	32.01 ± 16.08	27.10 ± 7.16
C18:2n6	1.28 ± 0.05	1.29 ± 0.08	1.29 ± 0.03	264.41 b ± 10.42	282.28 b ± 11.97	312.91 a ± 19.57
C18:2 cis	0.10 ± 0.03	0.14 ± 0.05	0.12 ± 0.04	21.03 ± 7.19	30.29 ± 11.81	29.90 ± 8.96
ΣC18:3 trans	0.12 ± 0.03	0.16 ± 0.05	0.16 ± 0.02	23.94 ± 7.22	35.36 ± 9.22	39.99 ± 6.24
C18:3n3 ALA	0.45 a ± 0.04	0.37 b ± 0.03	0.35 b ± 0.03	93.14 ± 8.45	81.82 ± 5.39	84.08 ± 9.90
C20:0	0.17 ± 0.04	0.14 ± 0.02	0.19 ± 0.05	34.85 ab ± 7.56	29.98 b ± 5.37	46.34 a ± 11.19
ΣCLA	0.48 ± 0.06	0.46 ± 0.11	0.44 ± 0.05	98.80 ± 11.39	99.59 ± 24.08	107.19 ± 16.67
C20:1c11	nd.	nd.	0.10 ± 0.01	nd.	nd.	23.28 ± 3.11
C21:0	nd.	nd.	nd.	nd.	nd.	nd.
C20:3n6	0.06 b ± 0.01	0.10 a ± 0.01	0.11 a ± 0.01	11.53 b ± 2.53	21.75 a ± 2.78	27.49 a ± 2.11
C20:4n6	0.11 ± 0.01	0.11 ± 0.05	0.14 ± 0.02	23.35 ± 2.39	23.74 ± 11.26	34.16 ± 4.70
C22:0	nd.	0.06 ± 0.01	nd.	nd.	13.38 ± 0.21	nd.
C23:0	nd.	0.11 ± 0.01	nd.	nd.	22.95 ± 3.41	nd.
C24:0	0.09 ± 0.01	nd.	nd.	16.02 ± 2.46	nd.	nd.
C22:5n3	0.09 ± 0.01	0.09 ± 0.01	0.08 ± 0.01	19.20 ± 0.42	20.42 ± 1.57	20.35 ± 1.25

0, 30, 60—days of dietary supplementation; nd.—not detected; ∑CLA—conjugated linoleic acid: sum of 18:2 c9,t11, 18:2 t9,c11, 18:2 c7,t9, 18:2 t7,c9, 18:2 c11,t13, 18:2 t11,c13; ^{a, b, c}—means in rows marked with different uppercase super-scripts differ significantly at p < 0.05.

. The fatty acid profile was dominated by SFA (65.88–66.36%), MUFA accounted for 27.76–28.51%, and PUFA 3.25–3.56%. The fatty acid profile of the cheese did not fully reflect that of the milk. Significant differences in the proportion of individual fatty acids were mainly found after 60 d of the modified diet and after taking into account the fat content of the product. The cheese made from the milk of cows fed a humic-mineral supplement contained significantly (p < 0.05) more SFA in 100 g, including SCSFA and LCSFA (17% each). They also contained more OCFA (by 17%) and BCFA (by 14%). These cheeses also contained significantly (p < 0.05) more MUFAs (by 19%), including acids with a cis configuration. There were no significant differences in the content of trans-configuration MUFA acids and PUFAs. However, within PUFAs, there was a significant increase in the content of those in the n6 group (from 299.29 to 360.81 mg/100 g), which resulted in an increase in the n6/n3 ratio (from 2.68 to 3.94). The content of DFA increased by 15%, while that of hypercholesterolaemic fatty acids (HSFA) increased by nearly 20%. The value of the TI index significantly increased, while there were no significant differences in the values of the h/H and AI indices.

The papers published so far present the influence of various natural additives in the feeding of cows on the fatty acid profile of cheeses made from their milk. The most common are fat sources such as flaxseed [39], olive oil [40], fish oils [39], algae [41], babassu coconut [42], or sources of phenolic compounds—olive cake [43] or dried grape pomace [44]. To the best of the authors’ knowledge, this is the first study on the effect of using humic-mineral substances from oxyhumolite in the feeding of cows on the fatty acid profile of cheese, so it is difficult to carry out a comprehensive discussion of the results obtained with the literature data.

4. Conclusions

The present study is the first on the effect of humic-mineral supplementation for cow feed on the nutritional value of milk fat in raw milk and the most important product as cheese. In summary, the addition of the humic-mineral substances from oxyhumolite to the ration of cows amounted to 100 g/cow/day, and significantly increased the fat content of the milk, and significantly affected the partial sums of the fatty acids profile and the nutritional fatty acid ratios and indices, both in the fat of the milk and the cheese made from it. However, from a nutritional point of view, it is difficult to unequivocally assess the changes as beneficial or not for the consumer. In light of current dietary recommendations, deterioration in the quality of milk fat was observed, which was associated with a higher proportion of HSFA, higher AI and TI values, and a lower h/H index and a lower proportion of DFA and MUFA. However, there was an increase in PUFAs, BCFAs, and SFAs, including SCSFAs, which, despite being classified as saturated fatty acids, are seen as nutritionally beneficial. The cheese showed an increase in the content (mg/100 g) of SFA (including SCSFA and LCSFA), OCFA, BCFA, MUFA, and DFA. Adverse changes also included a higher content of n6 group acids and a higher ratio of n6/n3, HSFA, and TI. The results obtained are promising, especially in terms of the natural origin of the supplement and its health-promoting properties, which is in line with the European Green Deal. However, depending on the source of humic-mineral substances, the dose used, and the time of administration, its effectiveness may be varied. Therefore, in order to unequivocally establish the effect of humic-mineral supplementation in cows’ diets on the nutritional quality of fat in milk and milk-based products, the research should be continued and extended to other parameters characterizing fat quality.