Production of β-galactosidase for lactose hydrolysis in milk and dairy products using thermophilic lactic acid bacteria

The aim of this work was to establish optimal conditions for the maximum production of β-galactosidase using an industrially suitable medium. Lactobacillus delbrueckii subsp. bulgaricus ATCC 11842 was cultivated in skim milk, whey and whey permeate basal media, supplemented with whey protein products, yeast extract or De Man–Rogosa–Sharpe (MRS) broth, at pH 5.6 and 43°C. All supplementations of the whey and whey permeate basal media resulted in the enhancement of the specific growth rates, rate of lactic acid production and β-galactosidase activity. However, unsupplemented skim milk gave the greatest rate of lactic acid production (3.50±0.269 mg lactic acid ml⁻¹ media h⁻¹) and the highest β-galactosidase activity (5.491±0.116 U activity ml⁻¹ media); far superior to the best whey-based medium supplemented with MRS (2.71±0.176 mg lactic acid ml⁻¹ media h⁻¹ and 3.091±0.089 U activity ml⁻¹ media, respectively). A technologically feasible approach for the reprocessing of the spent skim milk was tested and a conceptual process scheme is proposed.     

Technological characterization of lactic acid bacteria isolated from Armada cheese (a Spanish goats’ milk cheese)

Thirty-one strains of lactic acid bacteria (LAB) isolated from Armada cheese, Sobado variety, (eight strains of Lactococcus lactis subsp. lactis, four strains of Lactococcus lactis subsp. cremoris, two strains of L. lactis subsp. lactis biovar. diacetylactis, two strains of Leuconostoc mesenteroides subsp. mesenteroides, two strains of Leuconostoc mesenteroides subsp. dextranicum, five strains of Lactobacillus plantarum, six strains of Lactobacillus casei subsp. casei and two strains of Lactobacillus brevis) were screened for their acidifying capacity and enzymatic activity, that included the rapid API-ZYM system, the proteolytic activity, the amino-, di-, and carboxypeptidase activity and the caseinolytic activity. The strains of L. lactis subsp. lactis exhibited the highest acidifying and proteolytic activity. Lipase and esterase activity was practically non-existent for lactococci and lactobacilli; a certain esterase activity was observed among leuconostoc. The highest aminopeptidase activity was demonstrated by the cell-free extract (CFE) of some strains of L. plantarum, L. casei subsp. casei and L. mesenteroides subsp. dextranicum. The CFEs of L. lactis subsp. cremoris and L. lactis subsp. lactis possessed carboxypeptidase and dipeptidase activities, at levels depending on the strain. Appreciable caseinolytic activity was detected for the CFE of L. plantarum and those some lactococci.     

Probiotic lactic acid bacteria isolated from traditional Korean fermented foods based on β-glucosidase activity

This study was designed to isolate lactic acid bacteria (LAB) with β-glucosidase activity and probiotic properties from Korean fermented foods. Among nine isolates, four LAB strains had excellent survival rates at pH 2.5 with 0.3% (w/v) pepsin for 3 h and 0.3% (w/v) oxgall for 24 h. Four LAB strains did not produce β-glucuronidase and showed adhesion ability to HT-29 cells that was superior to that shown by the reference strain Lactobacillus rhamnosus GG. All four strains were sensitive to ampicillin, tetracycline, chloramphenicol, and doxycycline. These strains were identified as Leuconostoc mesenteroides H40, Lactobacillus plantarum FI10604, L. brevis FI10700, and L. perolens FI10842 by 16S rRNA gene sequence, respectively. It was found that L. perolens FI10842 produced the highest β-glucosidase activity (49.10 mU/mL). These results indicate that the four LAB strains could be used as potential probiotic. Especially L. perolens FI10842 could be used as a starter culture for fermentation.     

Antifungal activity of 2 lactic acid bacteria of the Weissella genus isolated from food

Abstract: In the present study, a total of 116 lactic acid bacteria (LAB) strains isolated from Mill flour and fermented cassava were screened for their antifungal activity. Three strains among 116 were selected for their strongest inhibitory activity against food molds. These 3 strains were Lactobacillus plantarum VE56, Weissella cibaria FMF4B16, and W. paramesenteroides LC11. The compounds responsible for the antifungal activity were investigated. The strains displayed an inhibitory activity against targeted molds at acidic pH. However, the influence of organic acids was rejected according to the calculated minimal inhibitory concentration (MIC). Antifungal compounds were investigated in the cell‐free supernatants and phenyllactic acid (PLA) was detected in different amounts with a maximal concentration for Lb. plantarum VE56 (0.56 mM). Hydroxy fatty acid, such as 2‐hydroxy‐4‐methylpentanoic acid, was also produced and involved in the inhibitory activity of Lb. plantarum VE56 and W. paramesenteroides LC11. Antifungal LAB are known to produce PLA and 3‐hydroxy fatty acids and other organic acids with antifungal activity. This short communication focuses on antifungal activity from Weissella genus. The antifungal activity was attributed to antifungal compounds identified such as PLA, 2‐hydroxy‐4‐methylpentanoic acid, and other organic acids. Nevertheless, the concentration produced in the cell‐free supernatant was too low to compare to their MIC, suggesting that the inhibitory activity was caused by a synergy of these different compounds. Practical Application: Antifungal LAB are interesting to prevent food spoilage in fermented food and prolong their shelf life. In this way, chemical preservatives could be avoided and replaced by natural preservatives.     

Identification and quantification of acetic acid bacteria in wine and vinegar by TaqMan–MGB probes

A Real-Time PCR (RT-PCR) assay was developed using TaqMan minor groove binder (MGB) probes for the specific detection and quantification of five acetic acid bacteria (AAB) species (Acetobacter pasteurianus, Acetobacter aceti, Gluconacetobacter hansenii, Gluconacetobacter europaeus and Gluconobacter oxydans) in wine and vinegar. The primers and probes, designed from the 16S rRNA gene, showed good specificity with the target AAB species. The technique was tested on AAB grown in glucose medium (GY) and inoculated samples of red wine and wine vinegar. Standard curves were constructed with the five target species in all these matrices. Quantification was linear over at least 5 log units using both serial dilution of purified DNA and cells. When this technique was tested in GY medium and inoculated matrices, at least 10²–10³ cells/ml were detected. To quantify low populations of AAB in microbiologically complex samples, a PCR enrichment including part of the 16S–23S rRNA gene ITS region was needed to increase the amount of target DNA compared to non-target DNA.     

Characterization and selection of autochthonous lactic acid bacteria isolated from traditional Iberian dry-fermented salchichón and chorizo sausages

ABSTRACT:  A total of 192 lactic acid bacteria were isolated from 2 types of naturally fermented dry sausages at 3 different stages of the ripening process in order to select the most suitable strains as starter cultures in dry-cured sausage manufacture according to their technological characteristics such as glucose fermentation, lactic and acetic acid production, and proteolytic, lipolytic, and antimicrobial activities. Identification of the isolates revealed that 31.2% were Pediococcus pentosaceus , 26.9% Lactococcus lactis , 18.6% Pediococcus acidilactici , 17% Lactobacillus brevis , and sporadic isolates of Leuconostoc mesenteroides , Lactobacillus plantarum , and Lactobacillus curvatus . Most of the strains did not produce gas from glucose and showed the capacity to produce lactic acid rapidly. Some 25% of the strains were able to degrade tributyrin (esterase activity), but none showed lipolytic activity against olive oil and pork fat. Only 3 strains of P. acidilactici showed weak proteolytic activity against myofibrillar or sarcoplasmic proteins. Also, the same strains showed antimicrobial activity against Listeria monocytogenes . Nine strains with the best properties were preselected and tested for biogenic amine production. The results showed that two of the strains, identified as P. acidilactici by polymerase chain reaction, had the potential to be further tested as starter cultures in pilot processing of Iberian sausages.     

Agar-immobilized basil–lactic acid bacteria bioproducts as goat milk taste-masking agents and natural preservatives for the production of unripened goat cheese

Goat milk cheeses have become popular recently; however, many consumers do not choose these products because they have specific sensory properties that are not acceptable to all consumers and the shelf life of the cheese is short. The concept of this work was to increase overall acceptability and shelf life of unripened goat milk cheese by using Ocimum basilicum and lactic acid bacteria (Lactobacillus plantarum LUHS135, Lactobacillus paracasei LUHS244, Pediococcus pentosaceus LUHS100, Pediococcus acidilactici LUHS29, and Lactobacillus brevis LUHS140) bioproducts (basil-LAB) immobilized in agar. A basil-LAB bioproduct could be a promising multifunctional ingredient for cheese manufacturing because it has a low pH, high LAB count, and high total phenolic compound content (after fermentation pH decreased by 25.4%, LAB count averaged 7.2 log₁₀ cfu/g, and total phenolic compound content increased by 30.9%). Use of different LAB in the preparation of basil-LAB bioproducts had a significant influence on cheese pH and hardness, and compared with cheese samples prepared with nonfermented basil, cheese samples prepared with basil-LAB bioproducts had, on average, higher pH (by 2.6%) and lower hardness (by 36.0%), similar to the control cheese (without basil). Overall acceptability of cheese was significantly influenced by the basil-LAB bioproduct immobilization process; in all cases, cheese samples prepared with fermented and immobilized basil-LAB bioproduct had better acceptability (5 points). After 120 h of storage, cheese samples prepared with basil-LAB bioproducts fermented with LUHS135, LUHS244 and LUHS140, no enterobacteria were found, and we detected strong negative and moderate negative correlations, respectively, of LAB count with enterobacteria count and yeast/mold count (r = ?0.7939 and r = ?0.4495, respectively). Finally, immobilization increased LAB viability in fresh goat milk cheese, which led to a reduction in enterobacteria and mold/yeast contamination during storage and an increase in overall acceptability compared with nonimmobilized basil-LAB. Therefore, basil-LAB bioproducts fermented with LUHS135, LUHS244, and LUHS140 strains can be recommended for preparing fresh goat milk cheese with extended shelf life and high acceptability.     

Effect of probiotic co-cultures on physico-chemical and biochemical properties of small ruminants’ fermented milk

Small ruminants' fermented probiotic milk is an alternative to fermented cows' milk, especially because of the monounsaturated/polyunsaturated fatty acid profiles. The technological and biochemical potential of Bifidobacterium and Lactobacillus co-cultures, with or without inulin, on goats' and ewes' milk was assessed. Microbial stability, lactose consumption, organic acid production, proteolytic parameters and conjugated linoleic acid (CLA) production in situ, were followed in ewes' and goats’ fermented milk (EFM and GFM, respectively) over 21 days at 4 °C; technological feasibility for probiotic fermented milk production was shown. In EFM, all co-cultures presented high viable cell numbers (>7.0 log cfu mL⁻¹) throughout storage, presenting faster acidification capacities and higher CLA isomer levels than in GFM. Inulin had no impact on probiotic growth, yet contributed to storage stability. CLA isomers and proteolysis indices were co-culture dependent traits: for example, co-culture of Bifidobacterium animalis B94 with Lactobacillus acidophilus L10 registered the best CLA-production in GFM.     

Extension of Tosèla cheese shelf-life using non-starter lactic acid bacteria

Six strains of non-starter lactic acid bacteria (NSLAB) were used to extend the shelf-life of the fresh cheese Tosèla manufactured with pasteurised cows’ milk. The acidification kinetics of three Lactobacillus paracasei, one Lactobacillus rhamnosus and two Streptococcus macedonicus were studied in synthetic milk medium. Lb. paracasei NdP78 and NdP88 and S. macedonicus NdP1 and PB14-1 showed an interesting acidifying capacity and were further characterised for growth in UHT milk and production of antimicrobial compounds. Lb. paracasei NdP78 and S. macedonicus NdP1 grew more than 2 log cycles in 6 h. Lb. paracasei NdP78 was also found to produce a bacteriocin-like inhibitory substance (BLIS) active against Listeria monocytogenes. The four NSLAB strains (singly or in combination) were used to produce experimental pilot-scale cheeses which were compared by a panel. The cheese manufactured with the mixed culture Lb. paracasei NdP78 - S. macedonicus NdP1 was the most appreciated for its sensory properties. The cheeses produced at factory-scale showed higher concentrations of lactobacilli (7.90 log CFU/g) and streptococci (6.10 log CFU/g), but a lower development of coliforms (3.10 log CFU/g) and staphylococci (2.78 log CFU/g) than control cheese (4.86, 4.89, 4.93 and 5.00 log CFU/g of lactobacilli, streptococci, coliforms and staphylococci, respectively) processed without NSLAB addition. The food pathogens Salmonella spp. and Listeria monocytogenes were never detected. The dominance of the species inoculated was demonstrated by denaturing gradient gel electrophoresis (DGGE), whereas strain recognition was evaluated by randomly amplified polymorphic DNA (RAPD)-PCR. From the results obtained, Lb. paracasei NdP78 and S. macedonicus NdP1 were able to persist during the storage of Tosèla cheese and their combination influenced positively the sensory characteristics and shelf-life of the final product.     

Influence of preprocessing methods and fermentation of adzuki beans on γ-aminobutyric acid (GABA) accumulation by lactic acid bacteria

The effects of pre-processes (immersing, germinating, and cold shock) and fermentation conditions of adzuki beans on γ-aminobutyric acid (GABA) accumulation using mixed cultures of Lactococcus lactis and Lactobacillus rhamnosus were investigated in this study. Among the preprocessing methods, cold shock treatment resulted in the highest observed GABA content (201.2 mg/100 g); a 150-fold increase compared to the non-treated adzuki beans. The LAB strains grew rapidly in cold-treated adzuki bean substrates and reached 10⁸ cfu/ml after 24 h of fermentation at 30 °C. After optimization, the GABA yield reached 68.2 mg/100 ml; a 20-fold increase compared to the non-fermentation yield. The viable cell counts of LAB remained above 10⁸ cfu/ml after 28 days of storage at 4 °C. Our results suggest that the combination of cold shock pretreatment and fermentation by LAB may be used for the preparation of adzuki beans with high GABA content, which can then be used as a natural resource of functional foods.     

Physical and nutritional conditions for optimized production of bacteriocins by lactic acid bacteria – A review

ABSTRACT

There has been an increasing debate about the use of synthetic chemical compounds and the consequences of their use in food preservation. In this context, the utilization of some natural compounds produced by bacteria, showing an inhibitory effect against microorganisms associated with food contamination, have gained attention as preservation technology. In order to improve the production and yield costs of bacteriocins, detailed studies are necessary to determine the conditions that allow an optimized production and extraction of bacteriocins from lactic acid bacteria (LAB). In this context, this article aims to discuss the information regarding the main factors that influence bacteriocin production by LAB. The biosynthesis of bacteriocins can be influenced by various culture conditions, such as the composition of the medium, pH, temperature and growth kinetics of the microorganisms. One of the limiting factors for the use of bacteriocins on a large scale in food preservation is the economic factor. In order for the production costs of bacteriocins to be reduced, making them attractive, it is necessary to know the optimum parameters of production, thus maximizing productivity and making costs more attractive.     

Effect of adzuki bean sprout fermented milk enriched in γ-aminobutyric acid on mild depression in a mouse model

This study focused on the ability of adzuki bean (Vigna angularis) sprout fermented milk, which is rich in γ-aminobutyric acid (GABA), to relieve anxiety and mild depression. A high-yield GABA-producing strain, Lactobacillus brevis J1, from a healthy cow was screened, and its physiological and probiotic properties were evaluated. The effect of adzuki bean sprout fermented milk was investigated in vivo in a chronic depression mouse model. The results showed that Lb. brevis J1 had excellent probiotic properties, grew well at low pH and 3% NaCl, and adhered to the surface of HT-29 cells. The GABA-enriched (241.30 ± 1.62 µg/mL) adzuki bean sprout fermented milk prepared with Streptococcus thermophilus, Lactobacillus bulgaricus, and Lactobacillus plantarum, and Lb. brevis J1 can reduce and possibly prevent mild depression-like symptoms in mice (C57/B6) by increasing social interaction and enhancing the pleasure derived from movement. The research revealed that the GABA_B-cyclic AMP-protein kinase A-cAMP-response element binding protein (GABA_B-cAMP-PKA-CREB) signaling pathway was related to the depression-like symptoms and that levels of 5-hydroxytryptamine, norepinephrine, and dopamine in the hippocampus of mice increased after treatment with the adzuki bean sprout fermented milk. Our results suggest that GABA-enriched dairy products have the potential to prevent or treat mild depression-like symptoms in mice, which suggests a new approach for a dietary therapy to treat chronic social stress.     

Transfer rate of α-linolenic acid from abomasally infused flaxseed oil into milk fat and the effects on milk fatty acid composition in dairy cows

The objectives of the present study were to evaluate the transfer efficiency of α-linolenic acid (ALA) from the abomasum into milk fat, its interaction with milk fat content and yield, and the relationship between ALA and C16:0 in milk fat. Three rumen-fistulated multiparous Holstein cows at midlactation were used in a 3×3 Latin square design. Treatments consisted of abomasal infusion of (1) 110mL of water/d (control), (2) 110mL of flaxseed oil/d (low flaxseed oil, LFO), and (3) 220mL of flaxseed oil/d (high flaxseed oil, HFO). Experimental periods were continued for 2 wk and fat supplements were infused abomasally during the last 7 d of each period. Average dry matter intake and milk yield were not affected by oil infusion. Milk fat and lactose content tended to be greater with flaxseed infusion compared with the control. Plasma ALA was 2.9- and 4.0-fold greater with LFO and HFO, respectively. The apparent transfer efficiency of ALA to milk was 44.8 and 45.7% with LFO and HFO, respectively. The C16:0 content in milk fat was decreased by 3.59 and 5.25 percentage units, whereas the ALA content was increased by 1.68 and 3.09 percentage units with LFO and HFO, respectively. Similarly, C18:2n-6 was increased by 0.95 and 1.31 percentage units with LFA and HFO, respectively, without changes in other fatty acids (FA). Total polyunsaturated FA was 4.4 and 2.7% lower in the HFO and LFO, respectively, than in the control. Furthermore, C16:0 content in the milk fat was reduced to a greater extent than the increase in ALA content, as a 1.68 and 3.09 percentage unit increase occurred in ALA compared with a 3.6 and 5.25 percentage unit decrease in C16:0 for LFO and HFO, respectively, such that a negative correlation existed between ALA and C16:0 (r=-0.72). In conclusion, abomasal infusion of flaxseed oil dramatically increased the ALA content in plasma and milk fat. Because the replacement of C16:0 with ALA and C18:2n-6 occurred without changes in other FA presumed to be synthesized de novo in the mammary gland, this suggests that the preformed C16:0 was replaced, rather than being caused, by an overall suppression of de novo FA synthesis in the mammary gland.     

A multiplex polymerase chain reaction for the identification of cows’, goats’ and sheep's milk in dairy products

A multiplex PCR able to identify cows’, goats’ and sheep's milk in dairy products was developed. Specific primers were designed in the mitochondrial 12s and 16s rRNA genes so as to generate fragments of different length.The assay was applied to 19 cheeses from the retail trade in order to verify the label statements. The multiplex PCR results were confirmed by PCR-restriction fragment length polymorphism.The proposed multiplex PCR represents a rapid and sensitive method applicable on a routine basis. In fact it enables to detect, in a single step, goats’, sheep's and cows’ milk in dairy products with a good sensitivity threshold (0.5%).     

Acid induced gelation of soymilk,comparison between gels prepared with lactic acid bacteria and glucono-δ-lactone

The objective of this work was to compare the gelation of soymilk particles induced by the acidification of a commercial starter culture with that resulting by addition of glucono-δ-lactone (GDL). Structure formation was followed using rheology, and the microstructure was observed by confocal microscopy. Acidification of lactic acid bacteria resulted in a higher gelation pH (pH 6.29 ± 0.05) compared to that of a gel induced by GDL (pH 5.9 ± 0.04). This difference was attributed to the longer time available for rearrangements of the soymilk particles in soymilk with starter cultures compared to the fast acidification by GDL. In spite of the earlier gelation pH, there were no observed differences in the final gel stiffness measured at pH 5.1, the value of tan δ, the frequency dependence and the linear viscoelastic range of the gels measured at the final pH. Microstructural observations also showed a similar protein network structure.     

Analysis of 27 antibiotic residues in raw cow’s milk and milk-based products – validation of Delvotest® T

Delvotest® T was evaluated for its capability at detecting residues of 27 antibiotics in raw cow’s milk and in some dairy ingredients (skimmed and full-cream milk powders). The kit was used as a screening tool for the qualitative determination of antibiotics from different families in a single test. Results delivered by such a method are expressed as ‘positive’ or ‘negative’, referring to the claimed screening target concentration (STC). Validation was conducted according to the European Community Reference Laboratories’ (CRLs) residues guidelines of 20 January 2010 and performed by two laboratories, one located in Europe and the other in Asia. Five criteria were evaluated including detection capability at STC, false-positive (FP) rate, false-negative (FN) rate, robustness and cross-reactivity using visual reading and Delvoscan®. STCs were set at or below the corresponding maximum residue limit (MRL), as fixed by European Regulation EC No. 37/2010. Four antibiotics (nafcillin, oxytetracycline, tetracycline and rifaximin) out of 27 had a false-negative rate ranging from 1.7% to 4.9%; however, it was still compliant with the CRLs’ requirements. Globally, Delvotest T can be recommended for the analysis of the surveyed antibiotics in raw cow’s milk, skimmed and full-cream milk powders. Additional compounds were tested such as sulfamethazine, spiramycin and erythromycin; however, detection at the corresponding MRL was not achievable and these compounds were removed from the validation. Other drugs from the sulfonamide, aminoglycoside or macrolide families not detected by the test at the MRL were not evaluated in this study. Regarding the reliability of this rapid test to milk-based preparations, additional experiments should be performed on a larger range of compounds and samples to validate the Delvotest T in such matrices.     

Aflatoxins and cyclopiazonic acid in feed and milk from dairy farms in São Paulo,Brazil

The occurrence of aflatoxins (AF) B₁, B₂, G₁, G₂ and cyclopiazonic acid (CPA) in feeds, and AFM₁ and CPA in milk was determined in dairy farms located in the northeastern region of São Paulo state, Brazil, between October 2005 and February 2006. AF and CPA determinations were performed by HPLC. AFB₁ was found in 42% of feed at levels of 1.0–26.4 µg kg^?1 (mean: 7.1 ± 7.2 µg kg^?1). The concentrations of AFM₁ in raw milk varied between 0.010 and 0.645 µg l^?1 (mean: 0.104 ± 0.138 µg l^?1). Only one sample was above the tolerance limit adopted in Brazil (0.50 µg l^?1) for AFM₁ in milk. Regarding CPA in feed, six (12%) samples showed concentrations of 12.5–153.3 µg kg^?1 (mean: 57.6 ± 48.7 µg kg^?1). CPA was detected in only three milk samples (6%) at levels of 6.4, 8.8 and 9.1 µg l^?1. Concentrations of aflatoxins and CPA in feed and milk were relatively low, although the high frequency of both mycotoxins indicates the necessity to continuously monitor dairy farms to prevent contamination of feed ingredients.     

Phytanic acid concentrations and diastereomer ratios in milk fat during changes in the cow’s feed from concentrate to hay and back

The phytanic acid content in milk correlates with the amount of green items in the cows’ feed. For this reason, the four-fold methyl-branched fatty acid has been suggested as a potential marker for the authentication of organic milk. In this study, we attempted to provide further support for this idea by studying the progression of the phytanic acid level and diastereomer ratio in milk fat by transition of the diets from high proportions of concentrate (typical “conventional” feed) to hay (typical “organic” feed in winter) and back to “conventional” feed. Milk samples taken from three cows were analyzed on both the phytanic acid concentration and diastereomer distribution. The cows were initially fed with “conventional” feed (ground feed with high portions (30–45?%) of concentrate), then the feed was changed within 1?week to 100?% hay (“organic” feed), and after ~6?weeks, the feed of two cows was changed back to the initial feed with concentrate (phase C_b). During the “conventional” feeding at the beginning of the experiment, the phytanic acid concentration was low (100–130?mg/100?g milk fat). When the feed was changed to hay (“organic” feed), the phytanic acid concentration immediately increased to a stable level of about 160?mg/100?g lipids. Changing back the feed to “conventional” feed, the phytanic acid concentration dropped immediately back below the value measured in the initial phase. Likewise, the SRR/RRR-diastereomer distribution of phytanic acid in the milk was an excellent indicator for the changes in the cows’ feed. While the SRR/RRR-diastereomer ratio was >1.5 during “conventional” feeding, it immediately decreased to equal amounts of both diastereomers when hay was supplied as feed. Abandonment of concentrate in conventional feeding increased the phytanic acid content but the SRR-diastereomer was still dominant and thus the SRR/RRR-diastereomer ratio was different to organic milk. Our results indicate that both parameters, i.e., the phytanic acid content and SRR/RRR-diastereomer ratio need to be measured for authentication of organic milk.