Umesh Kumar Shandilya

Impact of Milk Derived β-Casomorphins on Physiological Functions and Trends in Research: A Review

β-Casomorphins are a group of opioid peptides released during gastrointestinal digestion or food processing from the β-casein of milk protein. Consequently, milk can be divided into A1 and A2 “like” groups depending upon the presence or absence of proline or histidine at the 67th position of β-casein. A1 “like” milk is postulated to be a source of BCM-7 as histidine allows the cleavage at this position, while A2 “like” milk has proline that resists the hydrolysis. On one hand, BCM-7 has been implicated as a risk factor for cardiovascular diseases, type I diabetes, and neurological disorders. On the other hand, various physiological effects of these peptides have also been documented, i.e., secretion of mucus, increased activity of superoxide dismutase and catalase, increased levels of prolactin, and analgesic role. In addition, many evidences correlate these peptides with various immunological functions, such as development of innate immunity, lymphocyte proliferation and cellular immunity, role in autoimmune diseases, histamine release, and allergy. In conclusion, the role of β-casomorphins in physiological functions remains controversial and more research with improved diagnostic techniques is needed to unravel the mechanism and study physiological functions of β-casomorphins. Thus, health-related aspects of β-casomorphins (positive, negative, and immunological impacts) have been comprehensively reviewed in this article.

Probiotic Dahi containing Lactobacillus acidophilus and Bifidobacterium bifidum modulates immunoglobulin levels and cytokines expression in whey proteins sensitised mice.

BACKGROUND Cow milk allergy is the most common food allergy in children. So far, no effective treatment is available to prevent or cure food allergy. This study investigated whether orally administrated probiotics could suppress sensitisation in whey proteins (WP)-induced allergy mouse model. Two types of probiotic Dahi were prepared by co-culturing Dahi bacteria (Lactococcus lactis ssp. cremoris NCDC-86 and Lactococcus lactis ssp. lactis biovar diacetylactis NCDC-60) along with selected strain of Lactobacillus acidophilus LaVK2 and Bifidobacterium bifidum BbVK3. Mice were fed with probiotic Dahi (La-Dahi and LaBb-Dahi) from 7 days before sensitisation with WP, respectively, in addition to milk protein-free basal diet, and control group received no supplements. RESULTS Feeding of probiotic Dahi suppressed the elevation of whey proteins-specific IgE and IgG response of WP-sensitised mice. In addition, sIgA levels were significantly (P < 0.001) increased in intestinal fluid collected from mice fed with La-Dahi. Production of T helper (Th)-1 cell-specific cytokines, i.e. interferon-γ (IFN-γ), interleukin (IL)-12, and IL-10 increased, while Th2-specific cytokines, i.e. IL-4 decreased in the supernatant of cultured splenocytes collected from mice fed with probiotic Dahi as compared to the other groups. Moreover, the splenic mRNA levels of IFN-γ, interleukin-10 were found to be significantly increased, while that of IL-4 decreased significantly in La-Dahi groups, as compared to control groups. CONCLUSION Results of the present study indicate that probiotic Dahi skewed Th2-specific immune response towards Th1-specific response and suppressed IgE in serum. Collectively, this study shows the potential use of probiotics intervention in reducing the allergic response to whey proteins in mice.

Effect of thermal processing of cow and buffalo milk on the allergenic response to caseins and whey proteins in mice.

BACKGROUND Heat treatment is the most common method for reducing pathogen load, but it remains controversial in reducing the incidence of hyperimmune reactions. The aim of this study was to compare the allergenicity of caseins (CSN) and whey proteins (WP) of thermally processed cow and buffalo milk in a mouse model. Swiss albino mice were sensitised by intraperitoneal injections (administered in three doses at weekly intervals) of CSN or WP from cow or buffalo milk for the evaluation of humoral response and splenocyte stimulation index. RESULTS After 3 weeks of intraperitoneal stimulation of mice with milk proteins, the sterilised milk protein group displayed significantly lowered (P ≤ 0.05) serum IgG and IgE levels, while considerably increased cow milk protein-specific responses (IgE) were shown by proteins of pasteurised milk compared with those of raw milk. The stimulation index of splenocytes induced by CSN or WP of boiled and sterilised milk was also lower (P ≤ 0.05) than that of raw milk of both cow and buffalo. CONCLUSION The experiment showed that boiling and sterilisation of cow and buffalo milk clearly affect the allergenicity by decreasing the humoral and cell-mediated responses in mice. All results indicated that CSN and WP of sterilised milk are less allergenic than those of raw milk in mice.

Identification of internal control genes in milk-derived mammary epithelial cells during lactation cycle of Indian zebu cow

The present study aims to evaluate the suitability of 10 candidate genes, namely GAPDH, ACTB, RPS15A, RPL4, RPS9, RPS23, HMBS, HPRT1, EEF1A1 and UBI as internal control genes (ICG) to normalize the transcriptional data of mammary epithelial cells (MEC) in Indian cows. A total of 52 MEC samples were isolated from milk of Sahiwal cows (major indigenous dairy breed of India) across different stages of lactation: Early (5-15 days), Peak (30-60 days), Mid (100-140 days) and Late (> 240 days). Three different statistical algorithms: geNorm, Normfinder and BestKeeper were used to assess the suitability of these genes. In geNorm analysis, all the genes exhibited expression stability (M) values below 0.5 with EEF1A1 and RPL4 showing the maximum expression stability. Similar to geNorm, Normfinder also identified EEF1A1 and RPL4 as two of the most stable genes. In Bestkeeper algorithm as well, all the 10 genes showed consistent expression levels. The analysis showed that four genes, that is, EEF1A1, RPL4, GAPDH and ACTB exhibited higher coefficient of correlation to the Bestkeeper index, lower coefficient of variance and standard deviation, indicating their superiority to be used as ICG. The present analysis has provided evidence that RPL4, EEF1A1, GAPDH and ACTB could probably act as most suitable genes for normalizing the transcriptional data of milk-derived mammary epithelial cells of Indian cows.

Dietary supplementation of Butyrivibrio fibrisolvens alters fatty acids of milk and rumen fluid in lactating goats.

BACKGROUND Conjugated linoleic acid (CLA) isomers have high health amelioration potential and hence it is of great interest to increase the CLA content in dairy products. The present study was conducted to investigate the effect of administration of high CLA producing Butyrivibrio fibrisolvens In-1 on fatty acid composition of milk and rumen fluid in lactating goats. Four groups (n = 5) of lactating goats were assigned the following treatments: Control (C) (basal diet); T1 (basal diet + linoleic acid source), T2 (basal diet + suspension of Butyrivibrio fibrisolvens In-1, 10(9) CFU head(-1)) and T3 (basal diet + linoleic acid source + suspension of Butyrivibrio fibrisolvens In-1, 10(9) CFU head(-1)). RESULTS Rumen liquor and milk samples were collected on days 0, 15, 30, 60 and 90 of the experiment and linoleic isomerase enzyme (LA-I) activity and fatty acid profiles were elucidated. Major effects of treatments were seen on day 30 of the experiment. Total CLA content of rumen fluid increased (P < 0.05) by 218.72, 182.26 and 304% whereas total saturated fatty acid (SFA) content was lowered (P < 0.05) by 6.1, 4.44 and 9.55% in T1, T2 and T3, respectively, as compared to control. Vaccenic acid in groups T2 and T3 increased (P < 0.05) by 66.67% and 105.7% as compared to control. In milk, total CLA increased by 2.03, 1.61 and 0.61 folds in T3, T2 and T1, respectively. Total monounsaturated fatty acid and polyunsaturated fatty acid content increased (P < 0.05) in group T3 by 14.15 and 37.44%, respectively. CONCLUSION Results of the present study indicated that administration of B. fibrisolvens In-1 along with a linoleic acid (LA) source is a useful strategy to alter the biohydrogenation pattern in the rumen that subsequently decreased SFA content while increased CLA and unsaturated fatty acids in ruminants milk.

Matrix‐based three‐dimensional culture of buffalo mammary epithelial cells showed higher induction of genes related to milk protein and fatty acid metabolism

Demanding transcriptomic studies in livestock animal species could be replaced by good in vitro models mimicking the function of mammary gland. Mammary epithelial cells (MEC) are the functional unit of the mammary gland. Extracellular matrix is known to be a key factor providing normal homeostasis in three‐dimensional (3D) environment as important signals are lost when cells are cultured in two‐dimensional (2D) environment. The aims of this study were to establish a buffalo mammary epithelial cells (BMECs) in 3D culture using extracellular matrix and to determine whether such a 3D culture model has different expression pattern than 2D counterpart. The purified MEC generated after several passages were used to establish 3D culture using Geltrex matrix. The expression of milk casein genes viz., alpha S1‐casein (CSN1S1), alpha S2‐casein (CSN1S2), beta‐casein (CSN2), kappa‐casein (CSN3); and fatty acid metabolism genes viz., butyrophilin (BTN1A1), glycerol‐3‐phosphate acyltransferase (GPAM), fatty acid‐binding protein 3 (FABP3), and stearoyl‐CoA desaturase (SCD) was assessed in 3D culture in comparison to traditional monolayer culture using qRT‐PCR. Notable morphological differences were observed for BMECs grown in 3D culture in comparison to 2D culture. Morphologically, epithelial structures grown in Geltrex matrix (3D) environment showed enhanced functional differentiation in comparison to 2D culture. In 3D culture, lumen and dome‐like structures were formed by day 5, whereas polarized acinus‐like structure were formed within 15 days of culturing. The expression data showed higher mRNA induction of milk casein and fatty acid metabolism genes in 10‐day‐old 3D BMECs culture in comparison to 2D monolayer culture. The result suggests that 3D organization of epithelial cells has favorable effect on induction of milk and fatty acid metabolism‐related genes. Therefore, matrix‐based 3D culture of MEC that recapitulate the structural and functional context of normal tissues could provide a better in vitro model to understand the mammary gland functioning of buffaloes.

Different stimulating effects of caseins and whey proteins of processed cow and buffalo milk on lymphocyte proliferation in vitro

In this study, caseins and whey proteins after thermal processing of cow and buffalo milk (pasteurized, boiled, and sterilized) were evaluated for their effects on proliferation of mouse splenic lymphocytes with concanavalin A (ConA), lipopolysaccharide (LPS), and milk proteins. Swiss albino mice (3 weeks old) were sensitized by intraperitoneal injections of caseins or whey proteins (20 µg/200 µL of phosphate-buffered saline) with aluminum hydroxide (alum) as adjuvant, while the positive and negative control groups were sensitized with ovalbumin and normal saline, respectively. The in vitro experiments showed that boiling and sterilization of milk decreases proliferation of cultured lymphocyte with mitogens and milk proteins as well. On the contrary, an increase of proliferation index was observed after pasteurization. These results highlight the impact of thermal processing of caseins and whey proteins on the hosts specific and nonspecific immune responses. processing, milk proteins, allergenicity, splenocytes